Phytoplankton Analyzer
PHYTO-PAM-II
and
Phyto-Win 3
Software V 3
Principles of Operation
2nd Edition: Nov 2016
PhytoPamII_2.doc
 Heinz Walz GmbH, 2016
Heinz Walz GmbH  Eichenring 6  91090 Effeltrich  Germany
Phone +49-(0)9133/7765-0  Telefax +49-(0)9133/5395
Email [email protected]  Internet www.walz.com
Printed in Germany
MAY 2002
CONTENTS
1 Safety instructions ........................................................................ 1 1.1 General safety instructions .......................................................... 1 1.2 Special safety instructions ........................................................... 2 2 PHYTO-PAM-II ........................................................................... 3 2.1 Compact Version ......................................................................... 5 2.1.1 Spherical Micro Quantum Sensor US-SQS/WB .................... 8 2.1.2 Stirring Device WATER-S (optional) .................................... 9 2.1.3 Battery Charger MINI-PAM/L ............................................ 10 3 PhytoWin Software installation and Phyto-PAM-II setup ..... 11 3.1 Measure PAR Lists.................................................................... 13 4 First fluorescence measurements ............................................. 16 4.1 Principle of distinguishing between different groups of
phytoplankton ............................................................................ 21 5 Saturation Pulse Analysis .......................................................... 23 5.1 Measurements with Dark-Acclimated Samples ........................ 23 5.2 Measurements with Illuminated Samples.................................. 24 5.3 Fluorescence Ratio Parameters ................................................. 25 6 Features of PhytoWin Software ................................................ 30 6.1 Elements for system operation and display of instrument status33 6.2 Channels-window ...................................................................... 39 6.2.1 Ft, F, Fm’, Fm’-F, Y(II), Fv/Fm and Mean Value display... 39 6.2.2 Zero Offset and noise N(t) ................................................... 40 6.3 Algae-window ........................................................................... 42 6.4 Settings-window ........................................................................ 44 6.5 Slow Kinetics window .............................................................. 49 6.6 Light Curve window.................................................................. 51 6.6.1 Edit Light Curves ................................................................. 53 6.6.2 Light Curve Fit-parameters .................................................. 56 CONTENTS
6.6.3 Comments on Light curves .................................................. 62 6.7 Report Window ......................................................................... 64 6.8 Reference-window deconvolution and Chlorophyll
determination calibration values ............................................... 68 6.8.1 How to generate Reference Spectra ..................................... 72 6.9 Fluo Spec -window.................................................................... 74 6.10 Fast Kinetik - window .......................................................... 75 6.10.1 O-I1 Fit window .............................................................. 77 6.10.2 Parameters and Output of Fo-I1 Rise Analysis ............... 81 6.11 VIEW - MODE .................................................................... 82 6.12 Main Menu Bar .................................................................... 85 6.12.1 Options: ETR Parameter ................................................. 87 6.12.2 Al current/PAR list ......................................................... 88 6.13 Script files ............................................................................ 90 6.13.1 Data Management ........................................................... 91 6.13.2 Editing Tools ................................................................... 92 6.13.3 List of Script File Commands ......................................... 93 7 Technical Specifications ........................................................... 101 7.1.1 PHYTO-PAM-II Compact Version ................................... 101 7.1.2 System Control and Data Acquisition ................................ 102 7.1.3 Battery Charger MINI-PAM/L .......................................... 103 7.1.4 Spherical Micro Quantum Sensor US-SQS/WB ................ 103 7.1.5 Transport Box PHYTO-T .................................................. 104 7.1.6 Accessory Stirring Device WATER-S ............................... 104 8 Rechargeable battery ............................................................... 105 9 References and further reading .............................................. 106 10 Index .......................................................................................... 112 11 Warranty .................................................................................. 115 MAY 2002
CONTENTS
11.1 Conditions .......................................................................... 115 11.2 Instructions ......................................................................... 116 CHAPTER 1MAY 2002 SAFETY INSTRUCTIONS
1
1.1
Safety instructions
General safety instructions
1.
Read the safety instructions and the operating instructions
first.
2.
Pay attention to all the safety warnings.
3.
Keep the device away from water or high moisture areas.
4.
Keep the device away from dust, sand and dirt.
5.
Always ensure there is sufficient ventilation.
6.
Do not put the device anywhere near sources of heat.
7.
Connect the device only to the power source indicated in the
operating instructions or on the device.
8.
Clean the device only according to the manufacturer’s
recommendations.
9.
If the device is not in use, remove the mains plug from the
socket.
10.
Ensure that no liquids or other foreign bodies can find their
way inside the device.
11.
The device should only be repaired by qualified personnel.
1
CHAPTER 1
1.2
SAFETY INSTRUCTIONS
Special safety instructions
1.
PHYTO-PAM-II Multiple Excitation Wavelength Phytoplankton and Photosynthesis Analyzer is a highly sensitive
research instrument which should be used only for research
purposes, as specified in this manual. Please follow the
instructions of this manual in order to avoid potential harm to
the user and damage to the instrument.
2.
PHYTO-PAM-II employs high intensity LED-array light
sources which may cause damage to the eye. Avoid looking
directly into these light sources during continuous illumination
or saturation pulses.
3.
Do not cover the ventilation grille rear side of the instrument.
2
CHAPTER 2
2
PHYTO-PAM-II
PHYTO-PAM-II
PHYTO-PAM-II represents the progress of established WALZ
fluorometers dedicated to aquatic research like XE-PAM, WATERPAM, previous PHYTO-PAM and MULTI-COLOR-PAM. Choice
of several measuring light wavelengths at variable measuring light
settings (intensity and frequency) offered by XE- and 4-wavelengths
PHYTO-PAM are combined with the high time resolution of
MULTI-COLOR-PAM. Thus enabling classical PAM analysis,
deconvolution of phytoplankton and analysis of fast induction
kinetics.
The PHYTO-PAM-II incorporates a multi-color Chip-On-Board
(COB) LED Array featuring 5 measuring light colors and 6 colors for
actinic light illumination. The 440 nm, 480 nm, 540 nm, 590 nm,
625 nm measuring light facilitates online differentiation of 4
different pigment types therefor deconvolution of green algae,
cyanobacteria, diatoms/dinoflagellates and phycoerythrin containing
organisms like cryptophytes. As in the first generation PHYTO-PAM
deconvolution bases on fluorescence excitation reference spectra.
But for the first time these spectra are not instrument specific but
universal. Due to spectral calibration of PHYTO-PAM-II instruments
reference spectra can be shared between users and instruments.
The PHYTO-PAM-II user surface is based on the proven PhytoWinsoftware featuring classical PAM analysis like the estimation of the
effective photochemical quantum yield of PS II and the
determination of photochemical and non-photochemical quenching
parameters. For extended applications a special Fast Kinetics mode
of operation is added for measuring the wavelength-dependent O-I1
fluorescence rise kinetics upon onset of pulses of strong actinic light
information. Thus enabling evaluation of the functional absorption
cross-section of PS II, σPSII, determined by light color and the
3
CHAPTER 2
PHYTO-PAM-II
pigment composition of photosynthetic organisms (Klughammer and
Schreiber, 2015)
Two versions of the PHYTO-PAM-II Phytoplankton Analyzer are
available. A PHYTO-PAM-II Compact Version with integrated
emitter-detector unit and a Modular PHYTO-PAM-II Version
featuring different emitter-detector units connectable to a Power-andControl-Unit.
Both versions of the PHYTO-PAM-II are operated by PhytoWin-3
software.
4
CHAPTER 2
2.1
PHYTO-PAM-II
Compact Version
The PHYTO-PAM-II Compact Version is a lightweight and easily
portable instrument. The splash-proof cast aluminium housing
incorporates the control unit as well as all essentials of the emitterdetector unit: the optical unit, the measuring and actinic LED-array,
the photomultiplier-detector and the cuvette retainer.
Fig. 1: PHYTO-PAM Compact Version
Front side:
 Power switch and green power indicator lamp
 USB socket to connect external PC for instrument control
 Charge socket, to connect Battery Charger MINI-PAM/L
(100 to 240V AC)
 Fuse plug, containing 3.15 AT main fuse of internal power
circuit
5
CHAPTER 2

PHYTO-PAM-II
Light Sensor socket, to connect the Spherical Micro
Quantum Sensor (US-SQS/WB) for PAR list calibration.
Top:
6

Measuring Head with optical port for inserting sample
cuvette

PVC centering ring with o-ring sealing against the inner
wall of the Measuring Head, serving as a guide for the
cuvette and as an adapter for mounting the optional
Miniature Stirring Motor Water-S and the optional Spherical
Micro Quantum Sensor US-SQS/WB.

Cup-shaped perspex inset sealing against an inner o-ring of
the Measuring Head, thus protecting the opto-electronical
components from spilled water samples.

A green LED shows the status of the internal
Photomultiplier
Detector.
The
photomultiplier
automatically is switched off at excessive light impact.

Quartz Cuvette with 15 mm outer and 13 mm inner
diameter; height 46 mm.

Darkening Hood covering the part of the Measuring Head
protruding from the housing.

Photomultiplier Detector The Photomultiplier supply
voltage is automatically switched off when it sees too much
light (red status LED on housing lights up).

COB LED-Array The light emitting diodes (LED) are
densely arranged on a 10 x 10 mm area. PHYTO-PAM-II
provides 5 differently coloured measuring lights (440 nm,
480 nm, 540 nm, 590 nm, 625 nm), and 5 actinic light
CHAPTER 2
PHYTO-PAM-II
sources (440 nm, 480 nm, 540 nm, 590 nm, 625 nm); the
latter are complemented by white (440-640 nm) and far red
(735 nm) LEDs (for measuring light and actinic light spectra
see Fig. 2). To compensate for varying brightness of the
different types of light emission diodes, more LEDs are used
when the intensity of the individual LED is lower.
Fig. 2: Typical normalized emission spectra of PHYTO-PAM-II light
sources. Spectra are not corrected for spectral response of the
spectrometer.
7
CHAPTER 2
PHYTO-PAM-II
2.1.1 Spherical Micro Quantum Sensor US-SQS/WB
Fig. 3: Spherical Micro Quantum Sensor US-SQS/WB
The Spherical Micro Quantum Sensor US-SQS/WB is included in
the system package as calibration of the PAR-List is essential for
correct assessment of all PHYTO-PAM-II functions.
The US-SQS/WB device consists of a submersible spherical
quantum sensor, US-SQS/L, and a small amplifier unit which is
connected to the light sensor socket. The amplifier is tuned to yield
correct PAR readings within the automated PAR-List calibration (see
chapter 3.1) and needs to be set to 1x.
The 3.7 mm diffusing sphere of the US-SQS/WB sensor picks up
photosynthetically active radiation (PAR) with an angular response
error of less than ± 5% from -100° to 100° angle. Hence, the sensor
is ideally suited to measure light conditions in the suspension cuvette
where reflections and scattering results in randomization of light
direction. For stability of calibration, it is important to keep the
8
CHAPTER 2
PHYTO-PAM-II
diffuser clean. If needed the diffuser can be gently cleaned by using a
cotton tip applicator moistened with some ethanol.
The position of the light sensor is adjustable and will be determined
for PHYTO-PAM-II applications by a 15.3 mm spacer above the
sensor hood.
2.1.2 Stirring Device WATER-S (optional)
Fig. 4: Stirring Device WATER-S
WATER-S stirring device can be particularly
measurements of rapidly settling samples.
useful
for
A special adapter ring is provided for mounting the optional Stirring
Device WATER-S to the PHYTO-PAM-II Compact version.
WATER-S runs on a long life 3 V Lithium battery (size CR 123A). It
features an on/off switch and a potentiometer knob for stirring rate
adjustment. In the bottom center, a stirring paddle is mounted on the
9
CHAPTER 2
PHYTO-PAM-II
motor axis (via split brass-tube adapter). The disposable paddle can
be removed by gentle pulling. The other way around, a replacement
paddle can be mounted by pushing its cylindrical end all the way into
the holder. For replacement of the battery the housing has to be
opened by pulling the white and grey halves apart. Separation of the
two halves is facilitated by forcing gently a thin flat body into the slit
(like finger nail or thin screw driver).
It should be noted that at high photomultiplier gain the paddle of the
WATER-S will cause some increase of noise. This is due to the fact
that some measuring light is reflected from the paddle towards the
photodetector, such that the background signal is approximately
doubled and the electronic noise is correspondingly increased.
Furthermore, there is an increase of sample noise caused by the
movement of cells or cell groups.
2.1.3 Battery Charger MINI-PAM/L
The Battery Charger MINI-PAM/L is provided for recharging the
internal lead-acid battery (12V/2Ah) of PHYTO-PAM-II. It is
connected to the Charge-socket on the front panel of the Powerand-Control-Unit. The charger, which operates at input voltages
between 100 and 240V AC, features overload protection. Battery
voltage is displayed on the Settings-window of PhytoWin Software.
10
CHAPTER 3
INSTALLATION
3
PhytoWin Software installation and
Phyto-PAM-II setup
PhytoWin-3 and manual versions can be downloaded from Walz web
page
http://www.walz.com/downloads/overview.html.
The
PhytoWin-3 program needs be installed on the PC going to be used
in conjunction with the PHYTO-PAM-II. At the end of the guided
installation procedure a Phyto-PAM_3 folder is created on the PC
with Data-directories of three different types of Phyto-PAM
Measuring Heads (Phyto US, Phyto ED and Phyto Compact Unit).
Into these directories all measured data will be written. Next to these
directories a script folder, the Phyto.exe file and after definition of
the used measuring head, a Phyto.cfg is located within the PhytoPAM_3 folder.
After download double click the file Setup.exe
After start of Setup.exe the Install Wizard is called up.
This wizard guides through the installation, at the end of which the
PhytoPAM-3 folder will be installed on the PC.
Links to PhytoPAM-3 Folder and to
PhytoWin-3.exe are established on the
desktop.
11
CHAPTER 3
INSTALLATION
Connect PHYTO-PAM-II to computer via USB-cable and start
PhytoWin program by clicking the PhytoWin-3 icon on the desktop.
The Start-window is displayed, showing the number of the current
PhytoWin version.
Following query appears:
to search for Com-Port and enter Measure Mode
to view and analyse data acquired by the PHYTO-PAM-II
and start the program in the viewing mode.
to quit the program.
12
CHAPTER 3
INSTALLATION
In both software operation modes (Measure
Mode and Viewing Mode) the selection of
the applied measuring head is essential, as
each measuring head features individual
parameters (e.g. relating to photomultiplier
sensitivity) that are stored in separate Data
directories. These parameters are essential
for correct storage and analysis of the measured data. After definition
of the measuring head the actual program is started with the 5channels excitation window being displayed.
NOTE:
When a computer installs the PhytoWin-3 user software first time for
a selected PHYTO-PAM-II measuring head, the internal PAR list
needs to be measured before bringing into operation. Calibrations
of the PAR-Lists and a valid .par file in the measuring head folder is
essential for correct assessment of PHYTO-PAM-II functions.
The default.par file is only for startup and not suitable for functional
operation.
The features of the PhytoWin-3 user software described in the
following sections apply to all measuring heads. Exceptions are
indicated.
3.1
Measure PAR Lists
To bring the PHYTO-PAM-II to operation first time the LED array
light parameters (PAR lists) need to be calibrated. The light list
measurements include calibration of the light sensor offset and
13
CHAPTER 3
INSTALLATION
readout of the complete LED array done in an automated calibration
procedure.

Switch on PHYTO-PAM-II and start PhytoWin-3
software. Please plug in the Spherical Micro Quantum
Sensor US-SQS/WB to the PHYTO-PAM-II light sensor
connector, the light sensor will be recognized and the
Measure PAR Lists button in the Settings Window
enabled.
At first use the light sensor might show an offset. This
offset will be eliminated during light sensor calibration
within the Measure Par Lists routine.
14

Fill distilled water into the quartz glass cuvette and
insert it in the PHYTO-PAM-II measuring head.

Insert the light sensor. The position of the light sensor is
critical for correct detection of PAR values. Therefor the
light sensor/darkening hood geometry is fixed by a 15.3
mm spacer and should not be altered.

Open the measure PAR routine by clicking the Measure
PAR Lists button (Settings window) and start Light
calibration process.
CHAPTER 3
INSTALLATION

The light sensor offset and all LED array parameters will be
calibrated. When the Measure PAR List routine is finished a
.par file will be stored within the Data-directories of the
related measuring head folder.

Disconnect light sensor. The functionality of PHYTOPAM-II is established.
15
CHAPTER 4
FIRST MEASUREMENTS
4
First fluorescence measurements
Fig. 5
Channels-Window
The 5-channels excitation mode is the standard mode of operation of
the PHYTO-PAM-II. After start of the program, on the PC monitor
screen the "Channels"-window is displayed. This shows the current
Chl fluorescence yield, Ft, measured continuously with 5 different
excitation wavelengths. In addition, also the mean value of the 5
fluorescence signals is displayed. Normally, after program start the
displayed Ft-values are close to zero, as the Gain (photomultiplier
voltage) is set to a low setting by default, in order to avoid
unintended damage. As indicated by the status of the ML-switch
(bottom, left), the measuring light is switched on upon program start.
It is applied in LED-pulses, such that its actinic effect is relatively
weak. This means that no electrons accumulate at the acceptor side
of PSII and, hence, the minimal fluorescence yield, Fo, of a darkadapted sample is assessed. When the AL-switch is activated also
the intensity of the ML-LEDs is increased. This is due to an
automatic increase of the frequency of ML-pulses during actinic
illumination. In this way, the signal/noise ratio is increased and the
fluorescence changes induced by the actinic light are assessed. At the
16
CHAPTER 4
FIRST MEASUREMENTS
same time the ML at high frequency contributes to overall actinic
intensity, which is displayed in the PAR-field in units of
µmol quanta m-2s-1 (photosynthetically active radiation). A third type
of illumination is triggered by the "Sat-Pulse" button. But, please
avoid looking directly into the LED-array source, as this light is very
strong and may harm your eyes. This so-called "saturation pulse"
can cause complete reduction of the PSII acceptor pool and, hence,
induce an increase of fluorescence yield from its current level (Ft) to
its maximal value (Fm). Based on such measurements, the effective
quantum yield of photosynthetic energy conversion in PSII can be
determined, using the simple relationship:
Yield = (Fm-F)/Fm = Fm-Fo/Fm
For some first fluorescence measurements fill the cuvette with a
sample (which first may be pure water) and make sure that the
Photomultiplier-Detector is switched on (green indicator LED on
the right hand side of the elements of system operation bar).
Underneath the photomultiplier button is the Gain control box,
showing setting 5 of photomultiplier gain upon program start. This
gain is by far too low to show any fluorescence signal with a pure
water sample. After clicking the Gain-button the Gain-setting is
automatically increased until the channel with the largest signal
shows about 700 units (Auto-Gain function). Even pure water
samples will show a fluorescence signal, if the Gain is sufficiently
high (ca. setting 26 by Automatic Gain control). This unavoidable
"background signal" is due to stray fluorescence originating from
various system components like the LED-array, cuvette and filters.
You will find that an empty cuvette gives a much larger background
signal than a cuvette filled with pure water. The unavoidable
background signal can be digitally suppressed by the automatic
17
CHAPTER 4
FIRST MEASUREMENTS
Zero-offset function (Zoff). But, please note that it will always cause
a decrease in the signal/noise ratio.
In practice, natural surface waters often contain (besides
phytoplankton) other fluorescing substances (like humic acids) in
solution. In order to get rid of this contribution, together with the
small background signal caused by system fluorescence, it is
recommended to proceed as follows:
18

Make sure that the cuvette is clean, e.g. by washing with
ethanol and rinsing with water.

Make sure that the cuvette is placed correctly into the
Measuring Head. If the cuvette is not all the way down, this
will cause an increased background signal.

Fill the cuvette with ca. 4 ml of the sample to be
investigated and apply Auto-Gain to define the Gain-setting
at which the measurements will be carried out.

Prepare a filtrate of the sample using a 0.2 µm millipore
filter that will retain all phytoplankton.

Exchange the cuvette by a cuvette containing ca. 4 ml of the
filtrate and measure its fluorescence using the same Gainsetting as found appropriate for the unfiltered sample. By
giving a saturation pulse, you may convince yourself that
the signal displayed by the filtrate really is not originating
from active Chl. The Fm`-F and Yield-values will be zero or
close to zero. Please note the little indicator lamp below
the PAR-box, which lights up red as long as the signal is
unstable. All measurements, including Zoff-determination,
should be preferentially carried out after this lamp lights up
green. After Zoff-determination, the signals of the 5
channels are close to zero. Fluctuating values of up to ca. 5
units may occur due to digital noise and are of no concern.
CHAPTER 4
FIRST MEASUREMENTS
After Zoff-determination the filtrate is substituted by the sample and
now the proper fluorescence measurements can start, as the
fluorescence yields displayed for the 5 channels now are only due to
the phytoplankton. The most fundamental measurement is the
assessment of the quantum yield of photochemical energy
conversion in PSII by application of a saturation pulse. With an
active sample, the 5 channels will show values of maximal PSII
quantum yield (Yield under quasi-dark adapted conditions) in the
order of 0.5 - 0.8. You may have a look at the saturation pulse
kinetics (View Pulse check-box). For appropriate Yielddetermination, it is important that the maximal fluorescence yield is
reached during the saturation pulse, which is the case when a distinct
plateau is observed
Another fundamental measurement is the recording of the
fluorescence changes upon transition from darkness to continuous
light. Just switch on the actinic light (AL-button) and follow the
changes of fluorescence yield with time, Ft. You will observe that
fluorescence yield first rises to a peak level and then slowly declines
towards a steady state level. This is the famous Kautsky-effect,
which reflects the dark-light induction kinetics of photosynthesis. In
the slow kinetic window you can see the graphical displayed of the
recorded values.
When a saturation pulse is applied during actinic illumination, the
observed Yield-values are distinctly lower than after dark-adaptation.
This reflects a decrease in the efficiency of energy transformation at
PSII reaction centers due to two major factors: first, partial reaction
center closure (primary acceptor QA reduced) and, second, increase
of nonradiative energy dissipation.
Chl fluorescence carries information on the effective quantum yield
of PSII under quasi-dark and light conditions. The product of
quantum yield and quantum flux density of incident
19
CHAPTER 4
FIRST MEASUREMENTS
photosynthetically active radiation (PAR) provides a relative measure
of electron transport rate (ETR). Plots of quantum yield and ETR
versus PAR (so-called light response curves) give valuable
information on the photosynthetic performance and light saturation
characteristics of a sample.
The PhytoWin-program provides a routine for automated recording
of light response curves. For a first demonstration, open the "Light
Curve"-window and click the "Start"-button. There is an
immediate Yield-determination of the sample adapted to the
Measuring Light (at the given frequency of Measuring Light pulses).
Then light intensity automatically is increased in a first step (see
increase in displayed PAR-value) that extends over a defined time
period, at the end of which Yield again is determined. Further steps
of increased light intensity follow and at the end of each the Yield is
determined, thus resulting in light response curves of Yield and of
the derived ETR. The PAR-values of the various steps, the
illumination time during each step and the total number of steps can
be defined by the user (via Edit, see 6.6.1). These "Rapid Light
Curves" provide relevant information as outlined in more detail
below (see 6.6).
The fluorescence information obtained will be automatically stored
in the Report-file which can be accessed by clicking the
corresponding "register card" (Report see 6.7). All data stored in the
Report-file can be also recalled on the Channels- and Algaewindows for further inspection with the help of the VIEW-mode
(see 6.11). In order to continue with measurements, the user must
return to the MEASURE-mode.
20
CHAPTER 4
FIRST MEASUREMENTS
Principle of distinguishing between different
groups of phytoplankton
4.1
Well proven by the 1st generation PHYTO-PAM is the deconvolution
of different groups of phytoplankton. The reliable assessment of
fluorescence parameters using a number of different excitation
wavelengths is the basis for this distinction and characterization.
PHYTO-PAM-II features now a new spectral composition using five
excitation wavelengths that are chosen for optimal differentiation
between cyanobacteria, green algae, diatoms/dinoflagellates and
phycoerythrin-containing organisms, which differ substantially in the
absorbance spectra of their antenna pigments.
An essential prerequisite for the differentiation between various
types of phytoplankton is that the 5-channels fluorescence responses
of the pure cultures are known. The measurements of such
"Reference Excitation Spectra" are automated and, hence, quite
simple to be performed with the PHYTO-PAM-II, (see 6.7).
PHYTO-PAM-II normalizes measured reference spectra. Reference
spectra measured with PYTHO-PAM II are universal and can be
shared among users. More information about reference spectra are
given in chapter 6.8.
Based on the "Reference Spectra" the PhytoWin-program
deconvolutes the original 5-channels signals into the contributions of
the corresponding algal classes. It should be emphasized that
contrary to the unbiased fluorescence information displayed in the
"Channels"-window, the information on the "Algae"-window is
strongly biased by the information contained in the applied
References. Hence, the quality of the obtained results depends on
previous work invested by the user into the measurement of the
References. Such work will profit from background knowledge on
the likely presence of particular phytoplankton species in the
21
CHAPTER 4
FIRST MEASUREMENTS
investigated water sample. In this sense, the success of practical
applications to a considerable extent depends on close interaction
with basic research, not only using Chlorophyll fluorescence, but
also alternative methods, like microscopy, flow cytometry and
analysis by HPLC.
Deconvolution of the phytoplankton classes can be applied to data
obtained by classical PAM analysis as outlined in chapter 5.
22
CHAPTER 5
5
SATURATION PULSE ANALYSIS
Saturation Pulse Analysis
Typically, five different types of fluorescence levels are acquired by
Saturation Pulse analyses named Fo, Fm Fo’ Fm’ and F. In most
cases, the PAM fluorescence signal is proportionally related the yield
for chlorophyll fluorescence. Therefore, differences between these
five fluorescence levels reflect variations in chlorophyll fluorescence
yields.
Two of these levels (Fo and Fm) need to be determined with a darkacclimated sample. The three remaining levels (Fo’, F, and Fm’) are
repeatedly measured during subsequent sample treatments (e.g.,
exposure to actinic light; see Fig. 6).
5.1
Measurements with Dark-Acclimated Samples
Fo Minimum fluorescence level excited by very low intensity of
measuring light to keep PS II reaction centers open.
Fm Maximum fluorescence level elicited by a pulse of saturating
light (Saturation Pulse) which closes all PS II reaction
centers.
23
CHAPTER 5
Fig. 6:
5.2
SATURATION PULSE ANALYSIS
Measurements for Saturation Pulse Analysis. AL, Actinic
Light; D, dark; SP, Saturation Pulse; FR, Far-red illumination.
Measurements with Illuminated Samples
Fo’ Minimum fluorescence level of illuminated sample which is
lowered with respect to Fo by non-photochemical
quenching. When the measuring routine for Fo’ is active,
the Fo’ level is determined during a dark interval
following the Saturation Pulse. In the dark interval, farred light is applied to selectively drive PS I and to
quickly remove electrons accumulated in the intersystem
electron transport chain, thus reopening PS II reaction
centers (see Fig. 6, time point 75 s). Alternatively, the Fo’
24
CHAPTER 5
SATURATION PULSE ANALYSIS
can be estimated according to Oxborough and Baker
(1997):
Fo 
Fm’
F
5.3
1
1
1
1


Fo Fm Fm
Maximum fluorescence level of illuminated sample as
induced by a Saturation Pulse which temporarily closes
all PS II reactions centers. Fm' is decreased with respect
to Fm by non-photochemical quenching.
The F corresponds to the momentary fluorescence level (Ft)
of an illuminated sample measured shortly before
application of a Saturation Pulse.
Fluorescence Ratio Parameters
To quantify photochemical use and non-photochemical losses of
absorbed light energy, fluorescence ratio expressions have been
derived which are based on the relative fluorescence yields
introduced above. Table 1 compiles the fluorescence ratio parameters
available in PhytoWin-3. Subsequently, these parameters will be
briefly explained.
Fv/Fm and Y(II) Maximum and effective
quantum yields of PS II
photochemical
The Fv/Fm and Y(II) estimate the fraction of absorbed quanta used
for PS II photochemistry. Fv/Fm corresponds to the maximum Y(II).
For measurements of Fv/Fm, it is important that samples are
acclimated to darkness or dim light so that all reactions centers are in
the open state and non-photochemical dissipation of excitation
energy is minimal.
25
CHAPTER 5
Table 1:
SATURATION PULSE ANALYSIS
Fluorescence Ratio Parameters.
Source
Maximum photochemical quantum yield of
PS II (Kitajima and Butler, 1975)
Effective photochemical quantum yield of PS II
(Genty et al., 1989)
Quantum yield of light-induced (ΔpH- and
zeaxanthin-dependent)
non-photochemical
fluorescence quenching (Genty et al. 1996)*
Quantum yield of non-regulated heat
dissipation and fluorescence emission: this
type of energy loss does not involve the action
of a trans-thylakoid ΔpH and zeaxanthin
(Genty et al. 1996)*
Equation
FV FM  F0

FM
FM
Y(II) 
FM  F
FM
F F

FM FM
Y(NPQ) 
Y(NO) 
F
FM
Stern-Volmer
type
non-photochemical
fluorescence quenching (Bilger and Björkman,
1990)
NPQ 
FM
1
FM
Coefficient of photochemical fluorescence
quenching (Schreiber et al. 1986 as formulated
by van Kooten and Snel, 1990)
qP 
FM  F
FM  F0
Coefficient of photochemical fluorescence
quenching assuming interconnected PS II
antennae (Kramer et al. 2004)
qL  qP 
Coefficient of non-photochemical fluorescence
quenching (Schreiber et al. 1986 as formulated
by van Kooten and Snel, 1990)
qN  1 
F0
F
FM  F0
FM  F0
* Kramer et al. (2004) have derived more complex equations for Y(NO) and
Y(NPQ). Klughammer and Schreiber (2008) have demonstrated that the
equations by Kramer et al. (2004) can be transformed into the simple equations
of (Genty et al. 1996) which are used by Phyto-Win-3 software.
26
CHAPTER 5
SATURATION PULSE ANALYSIS
In algae and cyanobacteria, the dark-acclimated state often is not
showing maximal PS II quantum yield, as the PS II acceptor pool
may be reduced in the dark by stromal reductants and consequently
the so-called state 2 is formed exhibiting low PS II quantum yield. In
this case, preillumination with moderate far-red light should precede
determinations of Fo and Fm. The far-red preilluminated quasi-dark
state normally serves as reference state with maximal PS II quantum
yield for assessment of the functional absorption cross-section of
PS II, Sigma(II).
The Y(II) value estimates the photochemical use of excitation energy
in the light. It is lowered with respect to Fv/Fm by partial closure of
PS II centers and various types of nonphotochemical energy losses
induced by illumination. To derive from the Y(II) information on the
overall state of photosynthesis, knowledge of the absorbed PAR is
essential, as a sample e.g. may be severely damaged in Calvin cycle
activity and still show a high value of Y(II) in weakly absorbed light.
This aspect is particularly important in the study of algae and
cyanobacteria, which display large wavelength-dependent
differences in light absorption.
qP and qL Coefficients of photochemical fluorescence
quenching.
Both parameters estimate the fraction of open PS II reaction centers.
The qP is based on the concept of separated PS II antenna units
(puddle model), whereas the qL assumes interconnected PS II
antenna units (lake model) which appears to be the more realistic
situation in leaves (cf. Kramer et al., 2004). Determinations of qP an
qL do not require fluorescence measurements with the darkacclimated sample except when Fo’ is not measured but calculated
according to Oxborough and Baker (1997).
27
CHAPTER 5
SATURATION PULSE ANALYSIS
qN and NPQ Parameters of non-photochemical quenching
Both parameters are associated with non-photochemical quenching
of excitation energy, mainly involving a low thylakoid lumen pHand a zeaxanthin-dependent quenching mechanism. In contrast to
Y(II), qP and qL, calculations of the qN and the NPQ parameters
always require fluorescence measurements with the sample both in
the dark-acclimated and in the light-exposed states (see Table 1, page
26). Calculation of NPQ (or SVN; Gilmore and Yamamoto, 1991)
corresponds to the Stern-Volmer equation for fluorescence quenching
which predicts proportionality between fluorescence quenching
(NPQ) and the concentration of fluorescence-quenching centers in
the photosynthetic antennae (e.g. zeaxanthin).
Y(NO), Y(NPQ) and Y(II) Complementary PS II quantum
yields
Genty et al. (1996) first presented expressions based on basic
fluorescence parameters that describe the partitioning of absorbed
excitation energy in PS II between three fundamental pathways,
which were further investigated by Klughammer and Schreiber
(2004) and expressed in terms of the complementary quantum yields
of
Y(NO)
sum of non-regulated losses of excitation energy
including heat dissipation and fluorescence emission,
Y(NPQ) regulated energy losses of excitation energy via heat
dissipation involving ∆pH- and zeaxanthin-dependent
mechanisms, and
Y(II) use of excitation energy for charge separation.
The yields of photochemical energy conversion and nonphotochemical losses sum up to 1:
28
CHAPTER 5
SATURATION PULSE ANALYSIS
Y(II) + Y(NPQ) + Y(NO) = 1
This concept of "complementary PS II quantum yields" is useful to
analyze the partitioning of absorbed light energy in photosynthetic
organisms. For instance, under high light-conditions, a much higher
Y(NPQ) than Y(NO) indicates that excess excitation energy is safely
dissipated at the antenna level, that is, photosynthetic energy fluxes
are well-regulated. In variance, high values of Y(NO) would signify
that excess excitation energy is reaching the reaction centers,
resulting in strong reduction of PS II acceptors and photodamage,
e.g. via formation of reactive oxygen species.
29
CHAPTER 6 FEATURES OF THE SOFTWARE PHYTOWIN
6
Features of PhytoWin Software
Operation of the PHYTO-PAM-II Phytoplankton Analyzer is
based on the PhytoWin-Software in conjunction with a Pentium PC.
Installation of the software was described in chapter 3 and some first,
basic measurements using this software were already described in
chapter 4. Here the numerous functions supported by this software
are described systematically in some more detail.
The user surface of the PhytoWin-Software in the MEASURE-mode
is divided into 3 sections: 1. main menu, 2. one of nine output
windows and 3. system operation elements
Fig. 7: PhytoWin-3 software upon start
1) Main Menu: At the top, the items of the main menu are listed
(File, Window, Options and Help). Each item features a pull-down
sub-menu.
2) Window: The major central part of the screen features the
Channels-window, which represents one out of nine windows that
provide different ways of data output and user surfaces.
30
CHAPTER 6 FEATURES OF THE SOFTWARE PHYTOWIN
 Channels: Original, unbiased fluorescence information at
5 different excitation wavelengths
 Settings: Controls for instrument settings, like measuring pulse
frequency, actinic intensity, saturation pulse width and intensity,
clock interval, number of averages, etc.
 Algae: Deconvoluted fluorescence information for cyanobacteria,
green algae, diatoms/dinoflagellates and phycoerythrin-containing
organisms (e.g. cryptophytes)
based on previously recorded
reference excitation spectra;
 Slow Kinetics: Graphic display of saturation pulse analysis and
online recordings of Ft.
 Light Curve: Measurement and graphic display of light response
curves; effective quantum yield and relative electron transport rate
(ETR) as a function of PAR
 Report: File in which all measured data and instrument settings
are stored, which can be edited by the user and exported to other
programs
 Reference: Recording and display of reference excitation spectra
of cyanobacteria, green algae, diatoms/dinoflagellates and
phycoerythrin-containing organisms
 Fluo Spec: Display of measuring light characteristics and
unnormalized reference excitation spectra
 Fast Kinetics: Control and graphical display of fast kinetics
analysis providing information on the functional absorption crosssection of PS II (σII).
3) Elements for system operation and display: Below and at the
right hand side of the central output window a number of elements
31
CHAPTER 6 FEATURES OF THE SOFTWARE PHYTOWIN
for system operation and display of instrument status are located.
These elements always accessible, independently of the selected
window.
For handling of the PhytoWin-Software the standard Windows-rules
apply. For all possible operations "Tooltips" are provided which are
displayed whenever the cursor is moved into the vicinity of the
corresponding switch or button. They give a brief explanation which
in most cases should be sufficient even for an unexperienced user to
find his way through the program.
In the following sections some background information on the
different windows and instrument functions is provided.
32
CHAPTER 6.1
ELEMENTS FOR SYSTEM OPERATION
Elements for system operation and display of
instrument status
6.1
The elements for system operation and display of instrument status
are always available in MEASURE mode and independent of the
displayed window.
Fig. 8 User surface of PhytoWin-Software elements for system operation
and display of instrument status
Fig. 8 shows the user surface of the PhytoWin-Software in the
MEASURE-mode of operation with the Channels-window being
selected. Elements for system operation and display of instrument
status are marked red and will be briefly described starting from the
lower left corner and ending at the upper right corner:
On/Off switches of Measuring
Light (ML), Actinic Light (AL),
Far Red Light (FR) respectively.
33
CHAPTER 6.1
ELEMENTS FOR SYSTEM OPERATION
Display of current value of incident photosynthetically
active radiation (quantum flux density) within the cuvette
in units of µmol quanta m-2s-1. The displayed values are
either derived from an internal PAR-list or measured on-line with the
help of the Spherical Micro Quantum Sensor (see 0). If the light
sensor is connected the PAR: field turns green.
This indicator lamp may light up alternatively green or red.
When it lights up green, the system is ready for
measurement. This means that a stable reading is reached and the
signal/noise ratio is high. The indicator lamp lights up red after an
abrupt signal change (e.g. after switching on measuring or actinic
light or after changing photomultiplier gain) and whenever the signal
is disturbed by excessive background light.
ST = 50 µs single turnover saturation flash
AL + Y = actinic illumination with terminal
application of a saturation pulse for determination of
effective quantum yield (Yield = (Fm-F)/Fm). This function is active
only when the illumination time is defined (Act. Light Width on
"Settings"-window, see 6.4). Minimal Act. Light Width is 3 s.
Fo, Fm = determination of dark fluorescence yield of
a dark acclimated sample. Fo, Fm-determination is
essential for correct calculation of the quenching parameters.
Clicking this button automatically opens a new record, the
determined Fo, Fm values are stored in as measurement No. 1.
Fo,Fm values can be retained opening a new record marking the
checkbox “Keep Fo,Fm”.
34
CHAPTER 6.1
ELEMENTS FOR SYSTEM OPERATION
SAT-Pulse = Yield-determination by a single saturation
pulse. This measurement involves assessment of the
fluorescence yield briefly before the saturation pulse, F, and of the
maximal fluorescence yield, Fm’. While the Ft-values are
continuously changing and not stored, the F- and Fm’-values are
saved in the Report-file. The quantum yield of photochemical energy
conversion in PSII (Yield) is calculated by the equation:
Yield = (Fm’-F)/Fm’
The Yield-determination should be started only (via SAT-Pulse)
when any signal disturbance, has settled down, i.e. when the
indicator LED gives green light (see above).
Keep Fo, Fm: mark this checkbox to keep a measured Fo, Fm when
a new report or record is opened.
View
Pulse:
Check
box
to
display
the
fluorescence rise kinetics of the 5
signals during a saturation pulse.
This function is important to
ascertain that for a given sample the
settings of SAT-Pulse (intensity and
width) are appropriate to reach a
plateau of maximal fluorescence
yield during the pulse. This is a
prerequisite
for
proper
determination of effective quantum
yield.
35
CHAPTER 6.1
ELEMENTS FOR SYSTEM OPERATION
Start-button for Chlorophyll a content determination
based on fluorescence yield. Please note: the measured apparent
chlorophyll fluorescence is dependent on measuring light frequency
and needs to be done at the measuring light frequency chosen for
chlorophyll calibration during reference recording.
Script files are used for automated execution of
experimental procedures. The Load button opens up the
script file window for upload of an existing script file
or creation of a new script. To execute script files, click
on the Run button. More information about script files is given in
chapter 6.13.
Repetition Clock: When switched on
(check box), the selected command (Clockitem) is repeated indefinitely until manually
switched off again. The Clock interval
(time
between
two
consecutive
measurements) can be set (default value 20 s).
Minimal Clock interval is 3 s maximal clock
interval is 3600s. Please note that the Clock
items AL and AL+Y can be activated only
when AL Width has been defined under Settings (see6.4).
To leave the PhytoWin-program
In the MEASURE mode new data can be
measured and analysed online. The VIEW-mode
36
CHAPTER 6.1
ELEMENTS FOR SYSTEM OPERATION
is for offline inspection of previously stored data (see 6.11).
Manual start of a New Record, which is characterized
by the time and date of the moment at which New
Record was clicked. The records are subdirectories of a Report-file.
A New Record automatically is started upon start of the program,
starting a new slow kinetic, starting a new light curve or when
returning into the MEASURE-mode from the VIEW-mode
(see 6.11). It is recommended to start New Record manually with
every new sample or new experiment, and to write some relevant
information to the Record comment text.
Photomultiplier gain, which after start is by default
at the low setting of 5. When the Gain-button is
clicked, automatically the Gain-setting is adjusted to a value which is
suitable for measurements with a given sample (Auto-Gain
function). The gain steps are optimized to the extended dynamic
range of the PHYTO-PAM-II photomultiplier and comprises 30 gain
settings. The extended dynamic range distributes to the higher
sensitivity of PHYTO-PAM-II.
In the automatic gain (
) adjustment the maximal signal amounts
to about 700 units (Ft-value on Channels-window). The Gain can be
also manually adjusted using the arrows.
When the gain is too high the signal may run into “Overload” e.g.
during saturation pulses. A low gain may results in a too low signal
limiting the accuracy of fluorescence measurement. In both cases a
corresponding warning appears:
37
CHAPTER 6.1
ELEMENTS FOR SYSTEM OPERATION
Photomultiplier activation button. The indicator
lamp lights up green indicating an active
photomultiplier or red if photomultiplier is switched off.
Remaining illumination time during the course of an
actinic illumination period. The actinic illumination
period is defined on the Settings-window (Act. Light
Width). In conjunction with Light Curves (see 6.6), the width of each
actinic step is defined under Light Curves/Edit.
Date (and in View Mode also time) of the start of
the current record.
38
CHAPTER 6.2
6.2
CHANNELS WINDOW
Channels-window
The channels-window represents one out of nine windows which
can be selected for different modes of signal display and analysis, as
well as for definition of instrument settings.
The channels window displays the original, non-deconvoluted
fluorescence information at 5 excitation wavelengths.
Fig. 9 Channels-window for display of original fluorescence signals
measured with 5 different excitation wavelengths
6.2.1 Ft, F, Fm’, Fm’-F, Y(II), Fv/Fm and Mean Value
display
The current signals of the five different excitation channels are
displayed in the Ft-line and also by the five indicator-bars. Besides
the individual Ft-signals, also the Mean (average of the 5 signals) is
depicted.
After saturation pulse measurement (SAT-Pulse button) F, Fm’,
Fm’-F and Y(II) values are displayed for the five excitation
39
CHAPTER 6.2
CHANNELS WINDOW
channels. These values resemble the last stored values in the Report
window (see chapter 6.7). Also the dark adapted yield values of all
five excitation channels are displayed as Fv/Fm. If no measurement
is taken the corresponding value fields are crossed out
.
6.2.2 Zero Offset and noise N(t)
At low chlorophyll content, when high Gain is required,
the Ft-signals are not only due to Chl fluorescence but also
to an unavoidable background signal that originates from
various system components. Furthermore, in natural surface waters
fluorescing compounds like humic acids may be dissolved, which
will contribute to the signal. The contributions of such background
signals can be suppressed by the Zero Offset (Zoff) function.
To determine Zoff, the cuvette is filled either with pure water or with
the filtrate of a natural water sample. By clicking the Zoff button the
five background signals are measured and substracted from the
original Ft-signals, such that these are suppressed to zero. It is
recommended to determine Zoff at the same Gain as used for the
actual measurements (see also 3.1). Please note that the Zoff values
also are adjusted automatically to a changed Gain-setting on the basis
of the known Photomultiplier-Gain characteristic.
Please note: Activation of Zoff function initiates the immediate
measurement and subtraction of background signals. These Zoff
values remain valid as long as the Zoff function stays activated.
Measured Zoff values are noted in the report value, but not stored for
later applications.
40
CHAPTER 6.2
CHANNELS WINDOW
As an alternative to the display of the Zoff values, also
the momentary noise, N(F), on the individual
fluorescence channels can be shown. For display of
N(F), please click on N(F) in the selection box. When
the noise caused Fehler! Textmarke nicht definiert.by an external
disturbance has settled down, the indicator LED (below PAR-box)
gives green light for carrying out a measurement (e.g. application of
SAT-pulse). Please note that N(F) will include any time dependent
signal change, i.e. also time dependent fluorescence changes induced
by actinic light.
41
CHAPTER 6.3
6.3
ALGAE WINDOW
Algae-window
The Algae-window shows the deconvoluted fluorescence
information for cyanobacteria, green algae, diatoms/dinoflagellates
and PE-Type organisms (phycoerythrin-containing organisms e.g.
cryptophytes). The deconvolution is based on Reference Excitation
Spectra (see chapter 6.8) that were previously measured.
Fig. 10 Algae-window displaying deconvoluted fluorescence information
after application of a saturation pulse
In analogy to the Channels-window, the deconvoluted fluorescence
parameters Ft, F, Fm’, Fm’-F, Y(II) and Fv/Fm are displayed. In
addition also the deconvoluted Chlorophyll concentrations of the
four types of phytoplankton are shown.
In the same data boxes instead of Chl also the relative
electron transport rate, ETR (see chapter 6.12), or the
current noise, N(t) (see chapter 6.2.2), can be displayed.
Besides fluorescence yield, the indicator bars can
also show the Fm’-F induced by the last saturation
pulse or the Chl concentration. A corresponding
selection box is provided. Another selection box
42
CHAPTER 6.3
ALGAE WINDOW
allows the user to define maximal signal range for display at different
sensitivities. The F-bar shows the current fluorescence yield (Ft) in
the Measure- mode. In the View-mode the F-value sampled in
conjunction with the corresponding saturation pulse is displayed.
The Ft-values, as well as the corresponding indicator-bars, reflect the
online measured deconvoluted contributions of the four types of
phytoplankton to the overall fluorescence signal (i.e. the sum of the
5-channels signals). The sum of the four Ft-values displayed under
Algae and the sum of the five Ft-values displayed under Channels
are almost identical. The sum difference increases with increasing
"Fit error". The F-values correspond to the fluorescence yields
measured briefly before the saturation pulse and the Fm’-F values
represent the increases induced by the pulse.
43
CHAPTER 6.4
6.4
SETTINGS WINDOW
Settings-window
In the settings-window instrument settings are accessible for manual
adjustment.
Fig. 11 Settings-window PHYTO-PAM-II Modular Version
This button opens up a prompt for light
list calibration. This routine includes
calibration of the light sensor offset and determination of all
important characteristics of the LED array. Please read chapter 3.1.
Measuring Light Wavelengths: Five different
measuring light colors contribute to the measuring
light: 440 nm, 480 nm, 540 nm, 590 nm and 625 nm.
The contribution of each wavelength can be
inactivated by the corresponding checkbox. Please
note: For deconvolution of algae groups all five
measuring light wavelengths need to be activated.
44
CHAPTER 6.4
SETTINGS WINDOW
Measuring Light intensity: The measuring light
intensity can be set to High ML or Low ML
intensity by the pulldown menu. High light
measuring light intensities are required for
deconvolution.
Measuring Light Frequency: Meas. Freq. gives
the pulse frequency of the measuring light in
relative units. At the standard setting 2 the quantum
flux of photosynthetically active radiation
corresponds to ca. 1 mol quanta m-2s-1, which in
most cases is sufficiently low to allow assessment
of the dark-adapted fluorescence yield, which in
phytoplankton is not necessarily identical to minimum fluorescence
yield. The effective intensities of measuring light at different
frequencies are shown in the PAR-box.
Activate the Auto MF-High checkbox for
automatically increase of measuring light
frequency during actinic illumination and
saturation pulses. In the pulldown menu the
MF-H frequency can be set to 128, 32 or 8 Hz.
45
CHAPTER 6.4
SETTINGS WINDOW
Dial-boxes for Intensity and Width. A total of
20 intensity settings are provided. When Width
is set to 0 (default-setting), the actinic
illumination period is infinite and must be
manually terminated using the AL-button in the
elements for system operation bar at the bottom
left. At Actinic Light setting 0 (zero) no
separate Actinic Light is given, but measuring
light frequency is switched to maximal setting.
The actinic light color can be set to 440 nm,
480 nm, 540 nm, 590 nm, 625 nm or white via
pulldown menu.
Far Red light is adjustable in 20 far red intensity
levels with a width between 1 and 600 seconds.
When Width is set to 0 (default-setting), the far
red illumination period is indefinite and must be
manually terminated using the FR-Button in the elements for system
operation bar at the bottom left.
This function is only available for the PHYTOPAM-II / Modular version with a Miniature
Magnetic Stirrer PHYTO-MS connected and the manual stirrer
switch of the control unit in on position. Activation of the checkbox
activates the stirrer. Magnetic stirrer speed is adjustable by the
turning knob on the control unit front side.
Clicking the Default-button will restore the standard
settings. Settings changed by the user remain valid
after the program is left (via Exit) and started again at a later time.
46
CHAPTER 6.4
SETTINGS WINDOW
Display of present voltage of Power-and-Control-Unit
internal battery. When voltage drops below 11 V and the
blue indicator-bar disappears, the battery is almost empty
and needs to be charged via the provided battery charger.
There will be a warning: "Attention low battery! Please
connect battery charger."
Dial boxes for Intensity and Width of saturation
pulses. A total of 10 intensity settings is provided.
The pulse length (Width) can be varied between
40 ms and 500 ms. Default settings are 10 for
Intensity and 200 ms for the Width. Whether or
not Sat. Pulse Intensity and Width are appropriate,
may be judged for a particular sample with the help of the View
Pulse function (see chapter 6.1). The intensity and width of the
saturation pulse should be appropriate for the investigated sample
under the given experimental conditions. It must be assured that the
maximal fluorescence yield, Fm, is measured. The default setting of
saturation pulse intensity is 10 (maximal), as in most practical cases
errors are more likely to be due to the intensity being too low than
too high. An intensity which is sufficient to induce Fm after dark
adaptation, when electron transport is slow, may be too low after
light adaptation, when electrons are rapidly transported out of the
plastoquinone pool. While the maximal saturation pulse width is 500
ms, the default setting is at 200 ms, as in most samples a stable
plateau of Fm is reached within less than 200 ms. Furthermore, in
algae the fluorescence decline following the initial peak can be very
47
CHAPTER 6.4
SETTINGS WINDOW
fast. As Fm is determined from the average of data points at the end
of the saturation pulse, this may lead to underestimation of Fm, dF
and Yield.
The "View Pulse" function allows
to examine the fluorescence rise
kinetics during a saturation pulse. In
Fig. 12 the kinetics a saturation pulse
width of 0.3 s are displayed. In this
particular example, the fluorescence
yield rises to a plateau but also does
not declines during the saturation time
widths of 0.3 s. Saturation Pulse
settings are appropriate.
Fig. 12: View Pulse function for
assessment of rise kinetics
during saturation pulse
Clicking the New Report button will create a new
report file. The file will be created automatically
by the program, written into the Data-directories
of the applied Measuring Head. The file name is made up from the
date and number of report e.g. 20151117.rpt3 for the first report,
20151117_1.rpt for the second report started on the 17th November
2015. A report can include various record files characterized by the
time and date of the moment “New Record” was started.
48
CHAPTER 6.5
6.5
SLOW KINETICS WINDOW
Slow Kinetics window
The Slow Kinetics window graphically displays all data measured
by saturation pulse analysis. In addition by activation via start the
slow kinetic window provides online recording of current Ft values.
Fig. 13: Slow Kinetics window
Clicking the Start button initiantes a new record and
starts recording of current Ft values.
Clicking the Stop button terminates the slow kinetics
recording.
Ten different fluorescence parameters can be selected for
display. The display is controlled by checkboxes. Data
points and background colors of the corresponding
checkbox labels have identical colors.
49
CHAPTER 6.5
SLOW KINETICS WINDOW
It is possible to display either the Channels-data or
the deconvoluted Algae-data by selection via the
corresponding radio buttons.
On/Off switch for grid display
Prints a black/white copy of the graph for documentation
Autoscale option to display the complete graph
Courser position: left box displays x-axis
value, right box displays y-axis value.
Zoom in by clicking with the left mouse key on one corner of
the rectangular area to be magnified, keeping the key depressed
move to the opposite corner of the rectangle, and release key.

Shift y-axis by moving the mouse cursor vertically with the shift
key depressed.

The arrow keys in the lower right hand corner increase or
decrease the x axis range. Employing the arrow keys
deactivates the “Auto” function which otherwise automatically
adjusts the x axis for full display the fluorescence trace.
After Start of a Slow Kinetics a comment line will appear above the
graph. This comment line resembles the record comment of the
report window (chapter 6.7).
50
CHAPTER 6.6
6.6
LIGHT CURVE WINDOW
Light Curve window
Light curves give information on the light adaptation state and
photosynthetic capacity of a sample. With increasing quantum flux
density the effective quantum yield of PSII decreases, as PSII
reaction centers progressively close and an increasing amount of
energy is dissipated into heat.
Fig. 14 Light Curve-window showing the effective quantum yield of PS II
(Yield) and the relative electron transport rate (ETR) as a function of
incident PAR. Display of original Channels-data.
The Light Curve-window shows plots of effective quantum yield
(Yield) and relative electron transport rate (ETR) versus the incident
photosynthetic active radiation (PAR). The PAR-scale is
automatically adjusted to the selected range of light intensities,
which are defined by the Edit-subroutine (see below). By double
mouse-click on the Light Curves of Yield or ETR, either of these can
be displayed full scale. With another double mouse click
simultaneous display of both curves can be reinstalled.
51
CHAPTER 6.6
LIGHT CURVE WINDOW
It is possible to display either the
Light Curves of the Channels-data
(Yield and ETR measured with
five
different
excitation
wavelengths) or the Algae-data
(Yield and ETR of four different
pigment types of phytoplankton, deconvoluted using Reference
Spectra for Blue, Green, Brown and PE-type Algae). For selection of
Channels- or Algae-display the corresponding radio buttons are used.
Check boxes are provided to select the displayed channels or types
of phytoplankton. Deactivation of check boxes do not alter
deconvolution (in contrast to the check boxes on the
settings/reference window!).
If it is known that a sample does not contain a particular type of
phytoplankton, the corresponding reference should be inactivated at
the Reference-window level. In this way, the fitting error can be
reduced.
A Light Curve recording is started by clicking
the Start-button. A new record is started and
the recording follows the light curve protocol unless stopped via
Stop. Light curve protocols can be loaded and edited by the Edit
function (see chapter 6.6.1). The first measurement is made in the
absence of actinic illumination at the given Measuring Light
Frequency. Before light curve start Fo,Fm is determined for
parameter calculations.
During a Light Curve recording the number of
the current illumination step is indicated in
the Step-box. The remaining illumination time during a current step
52
CHAPTER 6.6
LIGHT CURVE WINDOW
is shown as Next: or in the AL Time-box in the elements for system
operation bar on the right hand side.
Light Curves can be auto scaled by clicking the autoscaleicon.
A Print-icon is provided for print-out of Light Curves via a
serial printer (please define printer settings in the main
menu).
In the main menu Options some
details in the way of Light Curve
presentation can be defined. Grid: For display of grid. Join: To
connect data points by line segments. For display of the Light Curve
Fit Parameters a separate window is opened, which is outlined in
sub-section 6.6.2.
6.6.1 Edit Light Curves
The Edit-function allows the user to define the
Intensity-settings and the time intervals of up to 20
steps in a light curve. Clicking the Edit button opens
a Light Curve Edit window (Fig. 15).
53
CHAPTER 6.6
LIGHT CURVE WINDOW
Fig. 15 Edit-window for definition of Light Curve parameters
For changing the Intensity-setting or Time (in units of 10 s) of a
particular step, the corresponding fields first have to be marked by
cursor/mouse click and then the desired setting can be typed in.
Steps
It is recommended to select progressively increasing PAR-values,
with the highest value being at least two times higher than the PAR at
which a sample was grown. Alternatively, it is also possible to edit
up-down light curves with light intensity first increasing to
saturation and then decreasing again. The observed hysteresis pattern
provides information on light activation parameters.
A light curve recording does not necessarily have to involve 20
illumination steps. When at a particular step under Time/10s a 0
(zero) is entered, the light curve is terminated with the preceding
step.
54
CHAPTER 6.6
LIGHT CURVE WINDOW
Intensity
One can choose between 20 intensity settings of actinic light and 8
frequency settings of the measuring light (MF1, MF2, MF4, MF8,
MF16, MF32, MF64 and MF128), which at higher frequencies has a
distinct actinic effect. The latter is relevant for assessment of the
early part of the light curve, where only a small decrease of Yield
occurs (or even a rise in some types of phytoplankton) and ETR
increases almost linearly with PAR. MF128 is equivalent to actinic
intensity-setting 0. Please note that there is a significant difference
between the spectral composition of the 5-wavelength measuring
light and the chosen actinic light.
PAR
The PAR indicated sums up incident PAR (PAR of the
corresponding intensity as listed in the applied PAR list plus PAR
resulting from the chosen Measuring Light frequency).
Time/10s
The Time-setting of each step can be set by typing units of 10 s (1 x
10 s up to 100000 x 10s) of a particular step.
Time/10s a 0 (zero) is entered, the Light Curve is terminated with
the preceding step.
The illumination period at each step is defined by
Time/10s. When "Uniform time" is activated, the
same time can be entered for all steps by typing a new value for one
step and confirmation by mouse-click.
55
CHAPTER 6.6
LIGHT CURVE WINDOW
Light Curve recordings may be started repetitively with the help of
the Clock-function (see 6.1). In this way, changes in the light
adaptation state and the physiological health of a sample can be
followed over longer periods of time.
The Light Curve Parameters can be saved in lcpfiles and reinstalled at any time by opening the
corresponding file. This provides great flexibility in Light Curve
recordings. An lcp-file applies to a particular type of Measuring
Head and is stored in the corresponding PhytoPam sub-directory
(Data_US6, Data_ED6 or Data_CU6).
Upon pressing the Default-button, a standard list of
intensity settings (with 9 steps ranging from MF64 to
AL7) and a uniform illumination time of 20 s are
installed.
After a previously defined list was changed, the
changes can be confirmed by OK or cancelled by
Cancel. In the latter case, the previously defined list of
Light Curve parameters is maintained.
6.6.2 Light Curve Fit-parameters
Light curve data can be analyzed using the EP model (Eilers and
Peeters (1988) Ecol Model 42: 199-215) or the Platt et al. model
(Platt T, Gallegos CL, Harrison WG (1980) J Mar Res 38: 687-701).
56
CHAPTER 6.6
LIGHT CURVE WINDOW
Rapid light curves plotting ETR/Yield versus PAR are often
described by the following three cardinal points.
α (alpha), electrons/photons: Initial slope of RLC which is related to
quantum efficiency of photosynthesis.
ETRmax, µmol electrons/(m2 · s): Maximum electron transport rate.
IK, µmol photons/(m2 · s): Transition PAR between light limited and
light saturated conditions.
The EP function used by the software has been derived by Eilers and
Peeters (1988) using a mechanistic model which considers the
processes of photosynthesis and photoinhibition. Their final model
function:
ETR 
PAR
a  PAR  b  PAR  c
2
In the above equation, the a, b, and c are free parameters which are
varied by PhytoWin until best fit between theory and data is
achieved.
The cardinal points of a light curve are calculated according to
subsequent equations:
α
1
c
ETRmax 
IK 
1
b  2 ac
c
b  2 ac
Fig. 16, illustrates the behavior of the Eilers and Peeters model
function for 3 theoretical cases which show identical values of α but,
at high light intensities, different degrees of photoinhibition.
57
CHAPTER 6.6
Cur
ve
Param
eter
a
b
c
α
ETR
max
Ik
1
1·10-5
1·1
0-2
5
0
.
2
41
2
0
7
2
1·10-6
1·1
0-2
5
0
.
2
69
3
4
5
3
1·10-7
1·1
0-2
5
0
.
2
88
4
3
8
Fig. 16:
58
LIGHT CURVE WINDOW
Exemplary Light Curves.
CHAPTER 6.6
LIGHT CURVE WINDOW
Also, the Platt et al. (1980) model considers photoinhibition but their
equation is not based on mechanistic considerations but on the
empirical finding the many light response curves can be described by
the product of 2 exponentials:
ETR  ETRmPot  (1  e

α  PAR
ETRmPot
)e

β  PAR
ETRmPot
The free parameters in the latter equation are ETRmPot, α and β,
where the ETRmPot is the asymptote value of the rising exponential in
the equation above. For correct fitting it is important to give initial
estimates for α and ETRmPot.
The initial fitting values for α result directly from slope of the initial
linear portion of the curve and ETRmPot is given by the highest
observed value of ETR.
The cardinal parameter β can be obtained with subsequent equation:
β
α
β α
ETRm  ETRmPot  (
)(
)
αβ αβ
IK 
ETRm

The model equation of Platt et al. (1980) considers photoinhibition
by the “photoinhibition parameter“ β. Platt et al. (1980) introduced
the “Photoinhibition Index” (Ib) to quantify photoinhibition. The
authors defined Ib as the PAR value required to photoinhibit ETRmPot
by the factor of 1/e according to:
Ib  ETR mPot β
59
CHAPTER 6.6
LIGHT CURVE WINDOW
The Fit button starts an iterative process during which
the free parameters of the selected model equations
(EP or Platt et al.) are varied until the best fit between theory and
experiment is obtained. The fitted curves are displayed as continuous
lines superimposed on the data points of the Yield- and ETR-Light
Curves. Table 2 summarizes the units of these parameters.
Table 2: LC Fit Parameters.
PAR
Fv/Fm x ETR fact
or
Estimated alpha
based on Fv/Fm.
alpha
Initial slope of light
curve.
electrons
quanta
ETRmax
Maximum electron
transport rate
µmol elect
m2  s
Ik
Photon flux at which
light-saturation of
photosynthesis sets
in.
µmol quan
m2  s
The original version of the model had to be modified in order to take
account of the fact that some types of phytoplankton (particularly
cyanobacteria) do not show maximal PS II quantum yield at PAR=0,
as assumed by the model, but at significant levels of PAR (ca. 20-40
µmol quanta m-2s-1). Hence, there is an initial rise and peak of Yield,
before the usual decline sets in at higher PAR values. This
phenomenon is likely to reflect a state 2 - state 1 pigment change.
The fitting routine applied by the PhytoWin program ignores the data
60
CHAPTER 6.6
LIGHT CURVE WINDOW
points in the rising part of the Yield Light Curve, including the peak
value.
A speed-button is provided for opening a window listing the
Light Curve Fit Parameters for Ch1-Ch5 and for the
deconvoluted types of phytoplankton (Blue, Green, Brown and PEType). The same window can be opened via the Main menu. The
parameter  (alpha): reflects the maximal slope of the ETR Light
Curve that, as outlined above, in phytoplankton samples is not
necessarily observed close to PAR=0. The numerical value of  is
equivalent to the maximal Yield multiplied by a PS II absorptivity
term. If relative ETR is determined, a value of 0.42 is assumed for
this term (ETR Factor multiplied by PS II distribution factor
(PPS2/PPPS.), in numbers: 0.84 • 0.5) For measurement of absolute
ETR the ETR Factor needs to be determined beforehand. The value
can be entered under Options/ETR Parameters (see 6.12 and 6.10).
ETRmax :represents maximal electron transport rate (relative units).
Ik corresponds to the particular PAR-value at the crossing point of the
lines defined by the initial slope (going through origin) and ETRmax
(parallel to PAR-axis). It is calculated from the expression /ETRmax.
Ik is characteristic for onset of light saturation.
61
CHAPTER 6.6
LIGHT CURVE WINDOW
Fig. 17 Light Curve Fit Parameters
The quality of Light Curves for Blue, Green, Brown and PE-type,
just like the deconvoluted values for fluorescence yield of Blue,
Green, Brown and PE-type (see section 6.3), strongly depends on
proper choice of Reference Spectra (see 6.7). Actually, in many cases
the noise introduced by the fitting may prevent a satisfactory
assessment of Light Curves for all three types of phytoplankton. This
will be particularly true for a component at low content, when
another type is dominating. The less Reference Spectra are used for
fitting, the lower will be the fitting noise. Hence, if e.g. it is known
from microscopic inspection that no or only few cyanobacteria are
present, the Blue reference spectrum should be inactivated, to
improve the results for green algae and diatoms.
6.6.3 Comments on Light curves
While in green algae, just like in green leaves, maximal PS II
quantum yield (Fv/Fm) is observed after dark adaptation (or
adaptation to low Measuring Light Frequency), this is not the case
with other types of phytoplankton. Particularly in cyanobacteria dark
adaptation leads to a state of low PS II excitation and low PS II
62
CHAPTER 6.6
LIGHT CURVE WINDOW
quantum yield (so-called state 2). In this case, maximal PS II
quantum yield is induced at moderate light intensities.
Despite similarity of Rapid Light Curves with classical light
response curves (P-I curves), there are also some basic differences,
which should be kept in mind when evaluating Light Curves. In
particular, the illumination time at each PAR-value generally is much
shorter for Light Curves than for P-I-curves. At the default setting of
20 s not sufficient time is given for the sample to reach a light
equilibrated state. Hence, such Rapid Light Curves (RLC) are
expression of the momentary light adaptation state of a sample. For
one and the same sample, there are as many different RLCs as there
are different states of light adaptation. The user may convince
himself about this fact by recording several consecutive RLCs
starting with a dark-adapted sample. With increasing number of
RLC-recordings, the ETRmax will increase: (unless there is
photoinhibition). On the other hand, there should be only one P-Icurve for a given sample when illumination times are sufficiently
long to allow full light adaptation at each intensity setting. In
practice, however, it is not always possible to keep a collected
sample physiologically healthy over longer periods of time (e.g.
limitation by insufficient CO2 supply must be avoided during such
long-term light curves).
63
CHAPTER 6.7
6.7
REPORT WINDOW
Report Window
The “Report” documents and saves automatically all experimental
data. A “Report” is organized in terms of separate “Records” with
information on type of experiment, number of saturation pulse
analyses, fast kinetics as well as the option to add comments. A
report and its associated records are automatically saved to the
Report folder of the corresponding PHYTO-PAM-II measuring head.
(e.g. C:\PhytoPam_DSP\ Data_CU6\Report).
In Measure Mode only the last Record can be analysed. In View
mode all Report/Record files are available for analysis.
Report:
New Reports can be started by clicking the New Report button in the
settings window or by clicking the
button in the Report window.
Report comments are entered in a text window which can be opened
by clicking on the Report comment icon
64
.
CHAPTER 6.7
REPORT WINDOW
Record:
A new record can be manually defined with the help of the New
Record button on the right-hand side of the elements for operation
bar.
A Light Curve or Slow Kinetics recording automatically start a new
record. Also upon program start and when returning to the
MEASURE-mode from the VIEW-mode, automatically a new record
is started.
It is recommended to enter a Record Title/Comment at the start of
each new Record. This comment can be written in the head line
available in the Record View of the Report window. This headline is
also available above the graphs in the slow kinetics and light curve
window. Additional information can be written in the text window
becoming available by the
button.
These radiobuttons switch between Report overview
giving information about the records (Time, Type,
numbers of saturaion pulse analyses, numbers of fast kinetic
measurements, number of chlorophyll concentration measurements,
and the record comment/title). A closer view on the data can be
obtained by selecting Rec (Record view). Please note in Measure
Mode only the current Record can be viewed. In the View Mode all
Records of the Report are available for detailed information and
analysis in the Rec view.
65
CHAPTER 6.7
REPORT WINDOW
The Record view (View Rec) provides detailed
information on an individual experiment for the last
record or in View Mode for all records. Minimum
number of column headlines in the data field is 6
(Date, Time, No.,Temp, ML, PAR). Columns for Fo´
fluorescence and fluorescence ratio parameters can
be added by selecting the item on the side bar by
ticking checkmarks in “Select Column”. The values
of Fo and Fm are listed in the F and Fm’ columns,
respectively. They are distinguished from other data
by column subtitles Fo and Fm.
Activation of this checkbox will state settings in the
record file. The abbreviations are G = Gain, MF =
Measure Light Frequency, AI = actinic light intensity, AW = actinic
light widths, FI = far red intensity, FW= far red widths, SI =
saturation pulse intensity and SW = saturation pulse widths. Also
setting changes within an experiment will be registered in the record
file.
Report-files can be analysed at any later time in the
VIEW-mode (see 6.11), which allows the user to try
out different Reference Spectra for minimizing the
fitting error etc. The Open Report and Export
Report commands apply to the VIEW-mode only.
In the VIEW-mode the Report-file can be exported into other
programs, like Excel or Sigma Plot for further analysis and different
forms of data presentation. The exported report comprises values
ticked with checkmarks in “Select Column” and will be saved as .csv
file. If not the complete Report but selected Record files are
supposed to be exported. The Export Record button
66
is used for
CHAPTER 6.7
REPORT WINDOW
export. The Report-file can be printed out, given that a suitable
serial printer is connected and has been defined by the Printer Setup
routine.
Export Record button exports the selected record file as .csv
file to the Export Folder of the corresponding measuring head. All
values ticked with checkmarks
in “Select Column” will be
exported.
67
CHAPTER 4.8
PHYTOWIN REFERENCE WINDOW
Reference-window deconvolution and
Chlorophyll determination calibration values
6.8
The primary signals measured by the PHYTO-PAM-II are five
different fluorescence signals obtained by excitation of the sample
with five measuring light beams at five different wavelengths (440,
480, 540, 590 and 625 nm). For the sake of deconvolution, the five
wavelengths were chosen to give optimal differences in the
excitation of the various antenna pigments that transfer excitation
energy to the Chl a in PS II. Each of the four main pigment types of
phytoplankton (cyanobacteria, green algae, diatoms/dinoflagellates
and Phycoerythrin containing organisms) is characterized by a
typical set of F-values at the five excitation wavelengths, which are
called the "Reference Spectra" or more shortly "References".
While they carry the information of five-point fluorescence
excitation spectra, these "spectra" also are shaped by the intensities
of the five differently colored excitation beams that on purpose are
not equal. The spectral composition of PHYTO-PAM-II instruments
are standardized by spectral calibration using the fluorescence
standard FluoRed and determined via the MEASURE PAR LIST
routine when the instrument was put to operation. Thus, contrary to
former versions of the PHYTO-PAM, PHYTO-PAM-II reference
spectra are universal.
Depending on conditions the fluorescence excitation spectra for the
fluorescence yield, F, and the saturation pulse induced increase, FmFo, can show significant differences. Therefore, the instrument
measures both F and Fm-Fo reference spectra and stores all relevant
instrument specific information within the reference spectra. The
instrument specific information allows normalization of the
references with respect to the spectral composition of the PHYTOPAM-II instrument. Therefore PHYTO-PAM-II reference spectra can
be exchanged between instruments and users.
68
CHAPTER 6.8
REFERENCE WINDOW
Additionally PHYTO-PAM-II references provide information about
chlorophyll content biased on fluorescence yield. Therefor each
reference spectrum includes chlorophyll calibration data which can
be set during reference generation (see 6.8.1).
All Reference information are stored in Ref6-files.
Fig. 18: Reference window Lists, numerical display of the five point
fluorescence excitation spectra
The "Reference Spectra" are defined as the normalized 5 channels
fluorescence signals of a particular sample displayed in the reference
window. Two Reference spectra views are available. The Lists view
or the Chart view.
In the lists view the Reference-window shows 2x5 Reference lines
with the data-boxes containing the numerical values of the
normalized 5-channels fluorescence signals F and Fm-Fo in different
colors (blue, green, brown and white). The corresponding data points
and connecting lines displayed in the chart view are blue, green,
brown and black. At the beginning of each reference line is a checkbox to activate or deactivate a particular Reference. Only when
69
CHAPTER 4.8
PHYTOWIN REFERENCE WINDOW
activated, the spectrum of a particular Reference is used for
deconvolution. If it is known, that a given sample does not contain
substantial amounts of a particular type of phytoplankton (e.g. green
algae in certain ocean waters), deconvolution of the other types of
phytoplankton will be improved by deactivating the corresponding
Reference.
Fig. 19 Simultaneous display of F and Fm-Fo Reference Specta in chart
view
Deconvolution consists in the fitting of the measured 5-channel
signals (F and Fm-Fo separately) by the sum of up to 4 components
(Blue, Green, Brown and PE-Type). The relative contribution of each
component to the 5-channel signals is determined by the individual
Reference Spectra. The sum of the squares of the deviations of the
measured data points from the fitted values is minimized (least
squares method).
The quality of fitting with a
particular set of Reference Spectra
can be judged by the Fit Error,
which is shown for the fluorescence yield, Ft, and for the saturation
70
CHAPTER 6.8
REFERENCE WINDOW
pulse induced fluorescence increase, Fm-Fo. The Fit Error depends
on the selection of References (via check-boxes) and on the choice of
the particular Reference Spectra files. If it is known that a sample
does not contain a particular type of phytoplankton (e.g. by
microscopic inspection), it is better to inactivate the corresponding
Reference.
A New Reference Spectra can be measured via the
New-button on the Reference-window. Reference
Spectra are measured with samples of pure cultures of phytoplankton
and comprise the wavelength dependent fluorescence characteristics
as well as the algae specific chlorophyll calibration. Instructions how
to generate reference spectra are given in chapter 6.8.1.
Together with the Reference a Comment file can be saved, in
which the type of sample and the conditions of Reference
measurement are documented. This Comment file can be readily
opened by clicking the speed-button in the corresponding Reference
line.
Previously saved References can be installed in the
corresponding Reference line by clicking the Load-button
and selecting the file name in the Data-directory of the applied
measuring head. Also references measured by other instruments can
be copied to the Data-directory of the applied measuring head.
Loaded references are normalized in respect to the spectral
composition of the PHYTO-PAM-II instrument.
71
CHAPTER 4.8
PHYTOWIN REFERENCE WINDOW
6.8.1 How to generate Reference Spectra
The quality of deconvolution bases on the quality of the applied
reference spectra. Reference spectra are generated from pure cultures
samples of algae. Data can be recalculated with new reference
spectra at any time. Also the reference spectra implemented
chlorophyll concentration calibration can be corrected manually at
any time after reference spectra generation.
72

Make sure your cuvette is clean. If necessary apply the
Zoff-determination using filtrate of sample to exclude
background fluorescence.

Prepare 2 ml of pure sample with a given concentration
of about 100 µg/l chlorophyll. The exact chlorophyll
concentration for chlorophyll calibration can be edited
manually e.g. after standard laboratory chlorophyll
determination.

Apply Auto-Gain to define Gain-settings at which the
measurement will be carried out.

Set the measuring light frequency. Keep in mind that
chlorophyll determination should be done at the same
measuring light frequency as chlorophyll content
calibration. Please set the measuring light frequency
during reference spectrum generation to the frequency
which will be used in following experiments.

Click
to start reference spectrum generation.
CHAPTER 6.8
REFERENCE WINDOW
A prompt to set chlorophyll
concentration
appears.
The
chlorophyll
concentration
is
predefined to 100 µg/l. The exact
chlorophyll concentration for
chlorophyll calibration can be
edited manually e.g. after standard
chlorophyll determination.

Finalize the reference spectrum by saving.
To edit the chlorophyll calibration implemented in the reference
spectrum please open the reference spectrum file in a text editor
program. The chlorophyll calibration data is listed by the value
Konz= (see Fig. 20) and can be changed manually to the chlorophyll
concentration determined with standard measuring procedures.
Fig. 20: Reference spectrum opened in a text editor for manual correction of
chloropyhll content calibration value
73
CHAPTER 4.8
6.9
PHYTOWIN REFERENCE WINDOW
Fluo Spec -window
PHYTO-PAM-II References are universal. The Fluo Spec Window
shows the rawdata of the reference spectra and some of the spectral
information used for normalization.
Fig. 21: Fluo Spec window displaying rawdata of universal Reference
Spectra
The ML Par displays
the measuring light values of ActP 440 nm, 480 nm, 540 nm, 590 nm
and 625 nm according to the internal light lists of the reference
spectrum measured instrument.
The radiobutton selects the reference for F,
Fm-Fo rawdata display below.
74
CHAPTER 6.10
6.10
FAST KINETICS WINDOW
Fast Kinetik - window
The fast kinetic window is provides analysis of the wavelength
dependend O-I1 fluorescence rise kinetics upon onset of pulses of
strong actinic light. The obtained data can be fitted in the O-I1fitting
window (chapter 6.10.1) for evaluation of the functional optical
cross-section of PS II, Sigma(II). Important prerequisites for analysis
of the rise kinetics are
a) Low chlorophyll content (< 300 µg/L for 440 nm excitation)
to avoid light gradients.
b) Oxidized PS II acceptor-side (QA and PQ-pool) achieved by
far-red background or far-red preillumination.
c) Accurate determination of the O- and I1-levels. Specifically,
the O-level is assessed briefly before onset of actinic
illumination and the I1-level is the fluorescence level reached
by a single turnover saturating flash (ST) at the end of the
illumination period (depicted as dot).
The Start button in the bottom right corner executes the
75
CHAPTER 6.10
FAST KINETICS WINDOW
fast kinetic light flashes and records the resulting fluorescence
kinetics.
When ST is checked, all actinic LEDs contribute to a
single turnover flash which then is particularly strong.
Selection of a color for measuring light automatically
sets actinic light to the same color. On the other hand,
changing the actinic light color does not affect
measuring light color so that all combinations of
measuring and actinic colors are available. Via
pulldown menu the intensity of ML and Act pulse can
be defined. The corresponding PAR value is indicated.
Point averaging leads to reduction in noise on the
cost of time resolution. Hence, point averaging is
equivalent to signal damping. The number of
averages can be defined by the pulldown menu Avg.
All measured fast kinetics will be averaged and
saved in one fast kinetic file. The pulldown menu pre defines a series
of fast kinetics before averaging is started e.g. to exclude “Fast
Kinetic” recordings at non-steady-state conditions. The New button
starts a new series of fast kinetic averaging.
The display of the fast kinetics graph can be changed. Zoom in
by clicking with the left mouse key on one corner of the rectangular
area to be magnified, keeping the key depressed move to the opposite
corner of the rectangle, and release key. Zoom out by clicking the
autoscale button above the chart.
76
CHAPTER 6.10
FAST KINETICS WINDOW
Displays the values for Fo and I1 of the
displayed Fast Kinetics recording
Pulldown menu to select a fast kinetic file for
graphical display
The Calc button provides access to the O-I1 Fit
window
6.10.1 O-I1 Fit window
The O-I1 Fit window serves for analysis of the O-I1 rise kinetics by a
special fitting routine. The routine was developed for determinations
of the functional optical cross-section of PS II, Sigma(II), which is
wavelength- and sample-specific. The routine is based on the
reversible radical pair model of PS II (Schatz et al., 1988, Biophys J
54: 397-405; Lavergne and Trissl, 1995, Biophys J 68: 2474-2492)
and takes into account reoxidation of the primary PS II acceptor QA
as well as energy transfer between PS II units (connectivity).
77
CHAPTER 6.10
FAST KINETICS WINDOW
The immediate result of an O-I1 Fit is information on the time
constant of the fluorescence rise. With information on the incident
PAR driving this rise (saved in the PAR Lists), the functional
absorption cross-section of PS II is calculated by the program.
Knowledge of Sigma(II) permits transformation of incident PAR (in
µmol quanta · m-2 · s-1) into the rate of quantum absorption per PS
II complex, PAR(II) (in quanta · PS II-1 · s-1). Then, the rate of
linear electron transport per PS II reaction center, ETR(II)
(electrons · PS II-1 · s-1) can be estimated according to
ETR(II)=PAR(II) · Y(II)/(Fv/Fm) where Y(II) and Fv/Fm are the
photochemical yields of PS II in the light-exposed and quasi-darkacclimated state (i.e. with weak far-red background light),
respectively.
One kinetic file (Fit) or if more
than one kinetic file is marked
several fluorescence kinetics
(Common) can be simultaneously
78
CHAPTER 6.10
FAST KINETICS WINDOW
analyzed by curve fitting. To this end, the kinetics of interest have to
be marked in the data list of the O-I1 fit window. Thereafter, the field
“Common” is available. A parameter selected in the Common box
will be identical for all marked curves. By assignment of “common”
parameters the reliability of the fitting analysis can be improved, if it
is unlikely that the enabled parameters vary significantly between
measurements. For example, the rate of QA- reoxidation is not
expected to be influenced by the color of light used for driving the
O-I1 rise. Hence, it makes sense to enable the rate constant “1.reox
Tau” when fitting O-I1 rise curves measured with various AL and MT
colors
Display in O-I1 Fit table for all fitted parameters the
+/- % signal variations required for +/- 10%
changes in fit error. Note that small signal
variations are indicative of a reliable fit and normally are written in
green. When written in red, a parameter limit was reached during the
fitting process. If you work with dense samples please activate the
dense sample checkbox.
J = Initial value (at start of O-I1 Fit) of the free
parameter J.
Tau (ms) = time constant of QA- formation (QA
reduction); initial value (at start of O-I1 Fit) of
free parameter Tau (ms).
79
CHAPTER 6.10
FAST KINETICS WINDOW
Reoxidation of QA- formed during the O-I1 rise
is taken into account. “With reoxidation” should
be disabled if QA- reoxidation cannot occur, e.g.
in presence of the PS II inhibitor DCMU. 1. reox
Tau (ms) = Time constant of QA- reoxidation by
QB (first QA reoxidation time constant). The 1.
reox Tau is varied during curve fitting.
The button starts O-I1 fitting. The results are compiled in
the O-I1 Fit data table. At the same time, original traces and
fitted curves are shown in the Fast Kinetics window. If the
experiment used a single turnover flash to elicited I1 level
fluorescence, a yellow sloped line describes the fluorescence decay
right after the flash. The I1 level fluorescence was determined by
extrapolating back the sloped line to the end point of the single
turnover flash which is 1.05 ms in the sample below. The latter
procedure is required because primary data acquisitioning was
switched off during the single turnover flash to avoid signal artifacts.
Cancels O-I1 Fit and closes the calculation window
Sets all parameters to default values
80
CHAPTER 6.10
FAST KINETICS WINDOW
6.10.2 Parameters and Output of Fo-I1 Rise Analysis
Table 3:
Adopted from: Schreiber et al. (2012) Photosynth Res 113: 127-144
Parameters
Definition
Free model parameters
J
Unit: dimensionless
Tau
Unit: s
Sigmoidicity parameter. J = p/(1-p), p is Joliot’s
connectivity parameter describing excitation
energy flux from a PS II to neighboring PS II.
Time constant of light-driven QA reduction.
Tau(reox) Unit: s
Time constant of QA- reoxidation.
Data input
Fo (or O) Unit: mV
Fluorescence in the dark-acclimated state.
Derived from initial fluorescence level of F0I1
rise.
I1
Maximum fluorescence elicited by a saturating
single turnover flash. Derived from backextrapolation of fluorescence decay after
saturating single turnover flash.
Unit: mV
PAR
Unit: µmol quanta·(m² · s)-1
Photosynthetically active radiation.
Derived parameters
k(II)=1/Tau
Unit: s-1
Sigma(II)λ=k(II)·(L·PAR)-1
Unit: nm2
Rate constant of light-driven QA reduction
Wavelength-dependent functional cross-section
of PS II
PAR(II)=k(II)=Sigma(II)λ·L·PAR
Unit: quanta·(PS II · s)-1
Rate of quantum absorption of PS II
ETR(II)=PAR(II)·Y(II)·(FV/FM)-1
Unit: electrons·(PS II · s)-1
Wavelength-dependent PS II-based electron
transport rate
Constant
L=6.022 · 1023 mol-1
Avogadro constant
81
CHAPTER 4.11
6.11
PHYTO WIN VIEW MODE
VIEW - MODE
The PhytoWin software can be used in two
different modes, the MEASURE-mode and the
VIEW-mode. While the MEASURE-mode
requires connection of the PC with the turned-on
PHYTO-PAM-II, for the VIEW-mode only the PC is required. The
MEASURE-mode is for data generation, the VIEW mode for Data
inspection, recalculation or export.
Data generated in the Measure Mode are automatically stored in
Report/Record files. In the VIEW-mode these data can be viewed as
originally recorded and saved in the Report-file or modified e.g. by
selection of different Reference files (see 6.8) or chlorophyll
calibration data within the references. In this way, older data can be
analysed on the basis of new information on the composition and
properties of a particular sample.
Reports can be opened via the Open Report
command of the Main Menu - File or via the
Open Report
button underneath Select Record.
The open report selection window (see below)
displays all report folder on the left hand side and
windows for direct view of the Report text and information about
Records-dates, -times, -types as well as the Record comments (of the
marked folder) for easy data assignment.
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CHAPTER 6.11
VIEW MODE
The opened Report-file is displayed on the Reportwindow. The Report is organized into Records,
defined by Date and Time, referring to the moment at
which a particular Record was started in the
MEASURE-mode.
The arrows allow to jump to the first or the last
Record and to move forward and backwards by
single steps in the Records.
The Records contained in a particular Report-file
are numbered. The number of the selected Record
(characterized by Date and Time) is shown, as well
as the total number of Records. Also the number of
83
CHAPTER 4.11
PHYTO WIN VIEW MODE
"Lines" which relate either to a measurement involving the
application of a saturation pulse or Chl determination is listed.
After selection of a particular Record, the data of the corresponding
measurement are selected to be viewed and analysed in detail on the
various display windows (Channels, Algae, Slow Kinetics, Report,
Light Curve, Reference, Fluo Spec or Fast Kinetics). Upon start
the first Line of the corresponding Record is displayed. The display
can be altered by marking other lines in the Record window. The user
may visualize this aspect by selecting a particular Record of interest
and calling up the Channels-window. With each jump on the Recorddisplay from one line to the next, there are corresponding changes in
the data displayed on the Channels-window. Fast Kinetics recordings
can be selected via drop down menu in the Fast Kinetics window.
As in the Measure Mode the selected References determine the
deconvolution of the measured data. In the View Mode Data can be
recalculated recording to the applied references.
Data selected in the VIEW-mode can be
exported in form of a csv-file (comma
separated values). In this form, the data can be
further analysed with a spread-sheet program
like Excel. To export a complete Report file please use the Export
Report command in the Main Menu File, to export single Record
please use the
Export Record button in the Report Window.
If the instrument is properly connected, the user can switch anytime
from the MEASURE- to the VIEW-mode and vice versa. Upon
return to the MEASURE-mode a new report is opened.
84
CHAPTER 6.12
6.12
MAIN MENU
Main Menu Bar
The Main Menu Bar contains pull down menus for File, Window,
Options and Help.
File
Open Report
Export Report
Print Report
Printer Setup
Exit
Window
Channels
Settings
Algae
Slow Kinetics
Light Curve
Report
Reference
Fluo Spec
Fast Kinetics
Options
L Curve Details

L Curve Fit Parameters
ETR Parameters
Help
Tooltips
Info
Hotkeys
AL-current/PAR List
File:

Open Report is only available in the VIEW MODE

Export Report is only available in the VIEW MODE and exports
the complete Report to the Export folder.

Print Report function is available in the VIEW MODE. Prints
report data under given printer settings.

Printer Setup to define printer settings
Window:

Provides access to Channels-, Settings-, Algae-, Slow Kinetics-,
Light Curve-, Report-, Reference-, Fluo Spec- or Fast Kineticswindow.
85
CHAPTER 6.12
MAIN MENU
Options:

L Curve Details to select graphical display of light curves.
Enables grid (Grid) or (Join) joining of data points with line
segments.

L Curve Parameter opens the light curve Fit Parameter Window
displaying the fit parameter α, ETRmax and IK of the Eilers and
Peeters or Platt et al model fit. This window is also available via
calculation button in the light curve window (more information
see 6.6.2).

ETR-Parameter opens the ETR-Parameter as outlined in
chapter 6.12.1.

Al current/PAR lists opens PAR list window where the actinic
light current and resulting PAR values are listed. This window
provides the function to save or load PAR lists.
Help:
 Activation of the tooltips checkbox gives access to short help
messages. The tooltips appear moving the courser close to functional
elements throughout PhytoWin-3 software.

Info provides information of the used software version.

Activation of the Hotkeys giving quick access to:
a = Algae window, c = Channels window, e = Report window, l =
Light Curve window, r = Reference window, t = Settings window
s = triggers a SAT pulse measurement
Hotkeys giving access to windows are underlined in the window
name, e.g. t = Settings
86
CHAPTER 6.12
MAIN MENU
6.12.1 Options: ETR Parameter
Relative ETR: When Relative ETR is selected, no attempt is made
to estimate the absolute rate of photosynthetic electron transport, as
no assumption is made on the absorption of the incident PAR. In the
case of dilute phytoplankton suspensions, it is clear that only a
minute fraction of the incident light is actually reaching the PS II
reaction centers where primary charge separation takes place.
Nevertheless it is informative to compare values of relative ETR of
various photosynthetic organisms at the same intensity of incident
light. Many researchers are familiar with ETR measurements in
higher plant leaves for which normally an absorptivity of 0.84 is
assumed.
For the sake of comparison the same fictive absorptivity may be
assumed for a phytoplankton suspension:
Relative ETR = Yield x PAR x PPS2/PPPS x ETR-Factor
PPS2/PPPS: Photons absorbed by PS II relative to photons absorbed by
all photosynthetic pigments, default value for leaves is 0.5
ETR-Factor: ratio of photons absorbed by photosynthetic pigments
to incident photons, default value 0.84
87
CHAPTER 6.12
MAIN MENU
It is assumed that half of the quanta of the incident PAR are
distributed to PS II, the quantum yield of charge separation of which
is measured via fluorescence. Maximum relative ETR-values
calculated in this way, range from ca. 30 µmol electrons m-2s-1 with
shade grown samples to ca. 150 µmol electrons m-2s-1 in the case of
high light grown samples, irrespectively of whether phytoplankton
suspensions or leaves (or any other photosynthetically active
organisms, like corals, sea grasses or lichens) are studied.
Estimate of absolute ETR: In order to estimate absolute ETRvalues, quantitative information on the absorption of the incident
PAR is required. Such information can be obtained from
measurements of the absorbance spectrum and the Actinic Light
spectrum (see e.g. Gilbert et al 2000. J Plant Physiol 157: 307-314).
Alternatively, the so-called PSII absorption cross-section can be
determined from the light saturation curve of the fluorescence
increase induced by a single turnover flash (Ley and Mauzerall 1982.
Biochim Biophys Acta 680: 95-106; Dubinsky et al 1986. Plant Cell
Physiol 27: 1335-1349). Under Options/ETR Parameters a value
for Absorption Cross Section (in m2/gChl) can be entered on which
the estimate of absolute ETR is based. The default value of 4.5
m2/gChl was proposed by Nicklisch A and Köhler J (2001)
Estimation of primary production with Phyto-PAM-fluorometry. Ann
Report Inst Freshw Ecol Inland Fish Berlin 13: 47-60.
6.12.2 Al current/PAR list
PHYTO-PAM-II functions depend on accurate calibration of the
LED array including the generation of an valid internal PAR list.
This is done via automated routine of the MEASURE PAR LIST
function on the settings side (see chapter 3.1). The Al current/PAR
88
CHAPTER 6.12
MAIN MENU
list menu in the menu bar opens the currently used PAR lists in a
separate window.
PHYTO-PAM-II provides six actinic
light colors. The pull down menu
determines the PAR list display of actinic
light color or actinic light pulse color
(ActP of the fast kinetic mode). Each
PAR list consists of three columns:
<Setting> is the setting number for the
Actinic Light intensity, <Current>
represents relative values for the LED
current and <PAR> lists values of PAR in
µmol photons/(m2·s).
The LED currents are optimized for algal
investigations. A double click on the
column heading Value gives access to
edit the current values. The maximum
current value is 500. Changes of
current values need recalibration of
the internal PAR List via MEASURE
PAR LIST routine – otherwise PAR values will be incorrect!
Button to open a PAR file
Button to save the PAR file
gives access to the PAR comment file to see or edit PAR list
comment text.
Button to export PAR list as .csv file
89
CHAPTER 6.13
6.13
PHYTOWIN SCRIPT FILES
Script files
Script files are used for automated execution of experimental
procedures. Particularly, script files are advantageous when the same
type of analysis needs to be repeated frequently and when
complicated protocols must be exactly reproduced.
Fig. 22: Script file window
Generally, all manual operations in the Phyto-Win software can also
be carried out under the control of a Script file. In addition, Script
files offer commands for time management, combinations of subprograms and conditional commands.
To access the script file window, click on the Load button on the
bottom bar of Phyto-Win. To execute script files, click on
the Run button below.
90
CHAPTER 6.13
PHYTOWIN SCRIPT FILES
6.13.1 Data Management
Four commands are provided for script file management:
New Script File Clears the script file window and
prompts for a new script file name.
Open Script File Opens a script file with name
format “filename.PRG”. The default directory for
script files is C:\PhytoPam_DSP\Script Files. No
other directories can be selected.
Save Script File Saves to C:\PhytoPam_DSP\Script
Files.
Script File Comment Displays a text-window for
notes on the current script file. The content of the
window is saved as text file with name
“filename01.TXT” which is associated with the script
file “filename01.PRG”.
91
CHAPTER 6.13
PHYTOWIN SCRIPT FILES
6.13.2 Editing Tools
Copy The command stores one or several lines of
the current script file in the clipboard. To execute the
copy command, select one or several lines using the
mouse cursor (Left-click once to pick one line. Hold
down Shift key and select first and last line of a series
of script file commands. Hold down Ctrl key to select
several scattered lines.) Click Copy icon. The selected
commands are now available for pasting within the
current or into another script file using the Paste
command.
Paste To paste previously copied commands, select
a line in the target script file and click the Paste icon.
The pasted lines will be written below the selected
line.
Insert Generally, “Insert” icons transfer commands
from the command box to the script file window. The
upper insert icon transfers commands from the box
“Program Control Commands”, the lower icon
transfers commands from the “General Commands”
box. Double click on the command to be transferred
is equivalent to the insert icons.
To insert a new command in the program list:
- Select the command to be inserted by mouse cursor
in the command box - Select by mouse cursor in the
script file window (right text window of the line
below which the command shall be inserted
- Click Insert button.
92
CHAPTER 6.13
PHYTOWIN SCRIPT FILES
Delete Select one or several commands to be deleted
(see above) and click icon. The command is
equivalent to pressing “delete” on the keyboard.
Undo Delete Reverses the last delete action.
Disable/Enable Disables command lines of the
current script file, or enables previously disabled
command lines. Disabled lines are printed in grey. To
execute the command, select line(s) in script file
window and click icon.
BACK Closes script file window. In case that a subprogram is displayed, the Back button returns to the
script file which calls the sub-program.
6.13.3 List of Script File Commands
The list of script file commands are divided into three sections. The
upper one contains commands controlling the progress of script files
(Program Control Commands). The middle panel contains the title of
nine groups of commands which are listed in the bottom panel.
Unchecking titles in the middle panels hides the corresponding group
of commands in the bottom panel.
93
CHAPTER 6.13
PARAMETER
PHYTOWIN SCRIPT FILES
COMMAND,
COMMENT
INPUT
Section 1: -- Program Control Commands --
94
Call
Execute another PhytoWin script file as a subprogram. A sub-program
can be displayed by
double-clicking an
existing calling line. Use
the Back button (  ) to
return to main script file.
Sub-programs may call
further sub-programs.
Script file name
(filename.prg)
Begin of
Repetition
Block
Mark the beginning of a
series of commands
(repetition block) which
is to be repeated.
Name of repetition
block
End of
Repetition
Block;
Loops =
Mark the end of a
repetition block. A
repetition block may
contain other repetition
blocks. Repetitions can
also be terminated
depending on the levels
of Ft or temperature (see
section “Condition
Commands” at the end
of this list).
Number of
repetitions
CHAPTER 6.13
PHYTOWIN SCRIPT FILES
PARAMETER
COMMAND,
COMMENT
INPUT
TimeStep
(s) =
Define the time interval
between the beginnings
of two consecutive
events; if the TimeStep
command is not
preceded by an event,
the time interval starts
with script file
execution. A series of
time steps forms a time
scale along which
actions can be performed
with defined time
pattern.
Time interval in
seconds
Wait(s)
Define the time interval
after the last command
has been completed and
next command is carried
out. Wait steps can be
placed inside of a
TimeStep interval
provided that the total
time required for
command execution plus
wait steps is shorter than
the time step interval.
Time interval in
seconds
95
CHAPTER 6.13
PHYTOWIN SCRIPT FILES
PARAMETER
COMMAND,
COMMENT
INPUT
Message =
Halt script execution and
display a message.
Clicking OK on the
message window or
hitting the Enter key
continues execution of
the script file.
Message title and
message text.
Comment
=
Insert a comment line in
the script file. A
comment line can be
written to the slow chart
title using the “Paste to
Slow Chart” command.
Comment text
Spacer
Insert an empty line after
the currently selected
line.
None
Exit
Terminate and exit script
file.
None
Section: -- General Commands --
96
New
Record
Start a new Record.
None
Start
Induction
Curve
Start recording of a slow
kinetics: equivalent to
the “Start” button on
slow kinetics window.
None
Stop
Induction
Curve
Stop recording of a slow
kinetics: equivalent to
the “Stop” button on
slow kinetics window.
None
CHAPTER 6.13
PHYTOWIN SCRIPT FILES
PARAMETER
COMMAND,
COMMENT
INPUT
Start Light
Curve
Start recording of a light
curve: equivalent to the
“Start” button on the
light curve window.
None
Clock
Start or stop the
repetitive trigger.
Check/uncheck
Clock
Time
Sets or modifies clock
interval.
Clock interval in s
(=s). Increment in s
to decrease (-s) or
increase (+s) the
current clock interval
Clock
Mode=
Selects the action to be
triggered by the clock.
Note that the drop-down
list for triggered events
differ between the SPAnalysis and Fast
Acquisition modes.
Select from dropdown list
Stirrer
modular version only:
Switch stirrer on and off.
Stirring is activated by
the stirrer switch on the
front plate of the control
unit.
Check on/off
Section 2: -- Determination Commands -Fo,Fm
Determine Fo and Fm
level fluorescence and
calculate Fv/Fm.
None
97
CHAPTER 6.13
PHYTOWIN SCRIPT FILES
PARAMETER
COMMAND,
COMMENT
INPUT
SatPulse/Fast
Kin.
Perform saturation pulse
analysis (in SP Analysis
Mode). Perform fast
kinetics (in Fast Kinetics
Mode).
None
Fo
Determine Fo level
fluorescence.
None
Section 3: -- Actinic Light Commands -AL
Switch actinic light
on/off.
Check on/off
FR
Switch far red light
on/off.
Check on/off
ST
Trigger single turnover
flash.
None
Section 4: -- Measuring Light
Commands -ML
Switch measuring light
on/off.
Check on/off
Section 5: -- General Settings --
98
Recording
Mode
Select type of slow
kinetics (manual,
induction, or induction
and recovery).
Drop-down list
Set Gain
Set or modify gain (1 to
10).
Gain setting (= #).
Increment for
decrease (- #) or
increase (+ #)
CHAPTER 6.13
PHYTOWIN SCRIPT FILES
PARAMETER
COMMAND,
COMMENT
INPUT
Keep
Fo,Fm
Off: Determine Fo and
Fm values at start of
each induction or light
curve. On: Use the
previous Fo and Fm
values for all subsequent
saturation pulse
analyses.
Check on/off
Section 6: -- Actinic Light Settings -AL Color
Select actinic light color.
Check one of 6
colors
AL Int
Intensity setting of
actinic light
1-20
FR Int
Intensity setting of far
red light
1-20
SP Int
Intensity setting of
saturation pulse
1-20
AL Width
Illumination time
interval for actinic light
0 – 600 s
FR Width
Illumination time
interval for far red light
0 – 600 s
SP Width
Illumination time
interval for actinic light
0 – 500 ms
Section 7: -- Measuring Light Settings -ML Freq
Set frequency of
measuring light
Check one of 8
frequencies
Section 8: -- Fast Kinetiks --
99
CHAPTER 6.13
100
PHYTOWIN SCRIPT FILES
PARAMETER
COMMAND,
COMMENT
INPUT
Start Fast
kinetics
Starts recording of a Fast
Kinetics. Equivalent to
the “Start” button on the
fast kinetics window
none
New
Average
Starts recording of a Fast
Kinetics average series
none
With ST
Fast Kinetics recording
includes single turn over
flash
Yes or No checkbox
With AL
Fast Kinetics recording
includes actinic light
illumination
Yes or No checkbox
Average
precycles =
Set number of fast
kinetics precycles
Number of Fast
Kinetics precycles
Curves to
Average
Defines the numbers of
fast kinetic recordings to
average
Number of Fast
Kinetics recordings
to be averaged
Average
Fast Kinetic recordings
are averaged
Check on/off
ActP
Set Intensity of ActP
1-20
ActP color
Set color of ActP
Check one of 6
colors
ML P
Set Intensity of
measuring light pulse
1-20
ML color
Set color of measuring
light
Check one of 5
colors
CHAPTER 7
TECHNICAL SPECIFICATIONS
7 Technical Specifications
Subject to change without prior notice
7.1.1 PHYTO-PAM-II Compact Version
General design: Metal housing for PHYTO-PAM-II Power-andControl-Unit including all opto-electronic components as well as the
measuring chamber for 15 mm Ø quartz cuvette WATER-K.
Chip-on-board multi-wavelength measuring light LED emitter:
440, 480, 540, 590, and 625 nm for pulse-modulated measuring light;
2 intensity settings; 8 settings of pulse frequency and 3 settings of
auto MF-high pulse frequency
Chip-on-board multi-wavelength actinic LED array: 440, 480,
540, 590, 625 and 420-640 nm (white) for continuous actinic
illumination, up to 1400 µmol m-2 s-1 PAR; fast kinetik flashes up to
7000 µmol m-2 s-1 PAR;
saturation
pulse
up
to
-2 -1
5000 µmol m s PAR,
Far-Red LED: peak wavelength 725nm
Signal detection: Photomultiplier detector based on Photosensor
Module H-10720 (Hamamatsu)
Standard detector filter: long-pass filter > 650 nm
Sockets: charge socket for Battery Charger MINI-PAM/L, input
socket for US-SQS/WB Spherical Micro Quantum Sensor, USB
socket
101
CHAPTER 7
TECHNICAL SPECIFICATIONS
Communication: USB 1.1, USB 2.0 and USB 3.0 compatible
User interface: Windows computer with PhytoWin-3 software
Power supply: Rechargeable sealed lead-acid battery 12 V/2 Ah;
Battery Charger MINI-PAM/L (100 to 240 V AC)
Dimensions: 29 cm x 30 cm x 20.5 cm (l x w x h), aluminum
housing with carrying handle and cuvette cover
Power consumption: Basic operation 1.3 W, ML +AL at maximum
output 3.7 W. Saturation Pulse at maximum intensity, 6.2 W
Weight: 4.42 kg (including battery)
Operating temperature: -5 to +40 °C
7.1.2 System Control and Data Acquisition
Software: PhytoWin-3 System Control and Data Acquisition
Program (Windows XP/Vista, Windows 7 and 8) for operation of
measuring system via PC. Advanced multi-wavelength analysis of
Chl fluorescence in suspensions down to 0.5 µg/l, including
measurements of slow and fast induction kinetics (Kautsky effect, OI1 fluorescence rise), deconvolution of major pigment types of
phytoplankton (green algae, diatoms, cryptophytes, cyanobacteria).
Higher sensitivity (e.g. 0.1 µg/l) can be obtained by signal averaging.
Saturation Pulse Analysis: Measured: Ft, Fo, Fm, F, Fo' (also
calculated), Fm'. Fast polyphasic rise kinetics (time resolution up to
10 µs). PAR using Spherical Micro Quantum Sensor US-SQS/WB.
102
CHAPTER 7
TECHNICAL SPECIFICATIONS
Calculated: Fo' (also measured), Fv/Fm, Y(II), qL, qP, qN, NPQ,
Y(NPQ), Y(NO) and ETR,
Fitting Routines: Fitting routine for fast fluorescence rise from 0 to
the I1 level to determine functional absorption cross-section of PS II.
Fitting routine for determination of the cardinal points α, Ik and
ETRmax of light curves.
Computer Requirements: Processor, 0.8 GHz. RAM, 512 MB.
Screen resolution, 1024 x 600 pixels. Interface, USB 2.0/3.0.
Operating system: Microsoft Windows Windows 7, 32 and 64 bit
7.1.3 Battery Charger MINI-PAM/L
Input: 90 to 264 V AC, 47 to 63 Hz
Output: 19 V DC, 3.7 A
Operating temperature: 0 to 40 °C
Dimensions: 15 cm x 6 cm x 3 cm (l x w x h)
Weight: 300 g
7.1.4 Spherical Micro Quantum Sensor US-SQS/WB
Design: 3.7 mm diffusing Plexiglass sphere coupled to integrated
PAR-sensor via 2 mm fiber, compact amplifier unit and special
holder for mounting on sample cuvette;
Connects to: PHYTO-PAM-II LIGHT SENSOR connector
103
CHAPTER 7
TECHNICAL SPECIFICATIONS
Cable length: 3 m + 0.5 m
Size: Sensor: diameter 1 cm, length: 11 cm; 15.2 mm spacer ring for
light sensor positioning; Hood: 3.4 cm diameter, 3.2 cm height;
Amplifier: 5 x 5 x 5 cm (w x l x h),
Weight: 175 g
7.1.5 Transport Box PHYTO-T
Design: Aluminum box with custom foam packing for PHYTOPAM-II and accessories
Dimensions: 60 cm x 40 cm x 34 cm (l x w x h)
Weight: 5 kg
7.1.6 Accessory Stirring Device WATER-S
Design: Miniature stirring motor in plastic housing with adapter to
mount on top of the PHYTO-PAM-II cuvette; equipped with
disposable perspex stirring paddle; self-contained unit featuring
long-life 3 V Lithium Battery; potentiometer for adjustment of
stirring rate
Dimensions: 80 mm x 50 mm x 30 mm (l x w x h)
Weight:
95 g (incl. battery)
Technical specifications are subject to change without prior notice.
104
CHAPTER 8
RECHARGEABLE BATTERY
8 Rechargeable battery
The Phytoplankton Analyzer PHYTO-PAM-II is equipped with a
rechargeable sealed-lead acid battery.
The life time is 1-3 years and it depends on the specific application.
A 10 °C rise of the temperature will decrease battery life by approx.
25%. Near the end-of-life the standby capacity of the battery will be
reduced. When this reduction becomes persistently, please replace
the battery.
The battery cannot be overcharged, when the battery charger
supplied with the instrument is used! Do not use any other battery
charger!
Never store the instrument with a discharged or partially discharged
battery! It is recommended to charge the battery every three months
during the storage period.

For optimum performance always recharge the battery
immediately after discharging!

Never leave the battery in a discharged stage!

Never short-circuit the battery terminals!
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9 References and further reading
 Agati G, Cerovic ZG, Moya I (2000) The effect of decreasing
temperature up to chilling values on the in vivo F685/F735
chlorophyll fluorescence ratio in Phaseolus vulgaris and Pisum
sativum: the role of the Photosystem I contribution to the 735 nm
fluorescence band, Photochem Photobiol 72: 75–84
 Baker NR (2008) Chlorophyll fluorescence: a probe of
photosynthesis in vivo. Annu Rev Plant Biol 59: 89–113
 Bernhardt K, Trissl H-W (1999) Theories for kinetics and yields
of fluorescence and photochemistry: how, if at all, can different
models of antenna organization be distinguished experimentally?
Biochim Biophys Acta 1409: 125-142
 Bilger W, Björkman O (1990) Role of the xanthophyll cycle in
photoprotection elucidated by measurements of light-induced
absorbance changes, fluorescence and photosynthesis in leaves of
Hedera canariensis. Photosynth Res 25:173-185
 Butler WL (1978) Energy distribution in the photochemical
apparatus of photosynthesis. Annu Rev Plant Physiol 29:345-378
 Dau H (1994) Molecular mechanisms and quantitative models of
variable PS II fluorescence. Photochem Photobiol 60:1-23
 Demmig-Adams B and Adams WW, III (1992) Photoprotection
and other responses of plants to high light stress. Annu Rev Plant
Physiol Plant Mol Biol 43:599-626
 Eilers PHC, Peeters JCH (1988) A model for the relationship
between light intensity and the rate of photosynthesis in
phytoplankton. Ecol Model 42: 199-215
 Franck F, Juneau P, Popovic R (2002) Resolution of the
Photosystem I and Photosystem II contributions to chlorophyll
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fluorescence of intact leaves at room temperature. Biochim Biophys
Acta 1556: 239–246
 Genty B, Briantais J-M, Baker NR (1989) The relationship
between the quantum yield of photosynthetic electron transport and
quenching of chlorophyll fluorescence. Biochim Biophys Acta 990:
87–92
 Genty B, Harbinson J, Cailly AL and Rizza F (1996) Fate of
excitation at PS II in leaves: the non-photochemical side. Presented
at: The Third BBSRC Robert Hill Symposium on Photosynthesis,
March 31 to April 3, 1996, University of Sheffield, Department of
Molecular Biology and Biotechnology, Western Bank, Sheffield, UK,
Abstract P28
 Genty B, Wonders J and Baker NR (1990) Non-photochemical
quenching of F0 in leaves is emission wavelength dependent:
Consequences for quenching analysis and its interpretation.
Photosynth Res 26: 133–139
 Gilmore AM, Yamamoto HY (1991) Zeaxanthin formation and
energy-dependent fluorescence quenching in pea chloroplasts under
artificially mediated linear and cyclic electron transport. Plant
Physiol 96: 635–643
 Govindjee (1995) Sixty-three years since Kautsky: Chlorophyll a
fluorescence. Aust J Plant Physiol 22:131-160
 Haldrup A, Jensen PE, Lunde C, Scheller HV (2001) Balance of
power: a view of the mechanism of photosynthetic state transitions.
Trends Plant Sci 6: 301-305
 Jakob T, Schreiber U, Kirchesch V, Langner U, Wilhelm C
(2005) Estimation of chlorophyll content and daily primary
production of the algal groups by means of multiwavelengthexcitation PAM chlorophyll fluorometry: performance and
methodological limits. Photosynth Res 83:343-361
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 Jassby AD, Platt T (1976) Mathematical formulation of the
relationship between photosynthesis and light for phytoplankton.
Limnol Oceanogr 21: 540-547
 Kitajima M, Butler WL (1975) Quenching of chlorophyll
fluorescence and primary photochemistry in chloroplasts by
dibromothymoquinone. Biochim Biophys Acta 376:105-115
 Klughammer C and Schreiber U (2008) Complementary PS II
quantum yields calculated from simple fluorescence parameters
measured by PAM fluorometry and the Saturation Pulse method.
PAM Application Notes 1: 27-35
(http://www.walz.com/e_journal/pdfs/PAN078007.pdf)
 Klughammer C, Schreiber U (2015) Apparent PS II absorption
cross-section and estimation of mean PAR in optically thin and dense
suspensions of Chlorella. Photosynth Res 123: 77-92
 Kramer DM, Johnson G., Kiirats O, Edwards GE (2004) New
flux parameters for the determination of QA redox state and
excitation fluxes. Photosynth Res 79: 209-218
 Krause GH and Weis E (1991) Chlorophyll fluorescence and
photosynthesis: The basics. Annu Rev Plant Physiol Plant Mol Biol
42:313-349
 Krause GH, Jahns P (2004) Non-photochemical energydissipation determined by chlorophyll fluorescence quenching:
characterization and function. In: Papageorgiou GC, Govindjee (eds.)
Chlorophyll a Fluorescence: A Signature of Photosynthesis. Springer,
The Netherlands, pp. 463-495
 Lazár D (1999) Chlorophyll a fluorescence induction. Biochim
Biophys Acta 1412: 1-28
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 Logan BA, Adams III WW, Demmig-Adams B (2007) Avoiding
common pitfalls of chlorophyll fluorescence analysis under field
conditions. Funct Plant Biol 34, 853–859
 Maxwell K, Johnson GN (2000) Chlorophyll fluorescence – a
practical guide. J Exp Bot 51, 659–668.
 Mohammed GH, Binder WD, Gillies SL (1995) Chlorophyll
fluorescence: A review of its practical forestry applications and
instrumentations. Scand J Forest Res 10:383-410
 Nedbal L, Koblížek M (2006) Chlorophyll fluorescence as a
reporter on in vivo electron transport and regulation in plants In:
Grimm B, Porra RJ, Rüdiger W, Scheer H (eds) Advances in
Photosynthesis and Respiration, Vol 25, Chlorophylls and
Bacteriochlorophylls: Biochemistry, Biophysics, Functions and
Applications. Springer, The Netherlands, pp 507-519
 Nicklisch A and Köhler J (2001) Estimation of primary
production with Phyto-PAM-fluorometry. Ann Report Inst Freshw
Ecol Inland Fish Berlin 13: 47-60
 Oxborough K, Baker NR (1997) Resolving chlorophyll a
fluorescence images of photosynthetic efficiency into photochemical
and non-photochemical components - calculation of qP and Fv'/Fm'
without measuring Fo'. Photosynth Res 54 135-142
 Peterson RB, Oja V, Laisk A (2001) Chlorophyll fluorescence at
680 and 730 nm and leaf photosynthesis. Photosynth Res 70: 185–
196
 Pfündel EE (1998) Estimating the contribution of Photosystem I
to total leaf chlorophyll fluorescence. Photosynth Res 56: 185–195
 Pfündel EE, Ben Ghozlen N, Meyer S, Cerovic ZG (2007)
Investigating UV screening in leaves by two different types of
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portable UV fluorimeters reveals in vivo screening by anthocyanins
and carotenoids. Photosynth Res 93. 205-221
 Pfündel EE, Klughammer C, Meister A, Cerovic ZG (2013)
Deriving fluorometer-specific values of relative PSI fluorescence
intensity from quenching of F0 fluorescence in leaves of Arabidopsis
thaliana and Zea mays. Photosynth. Res. DOI 10.1007/s11120-0129788-8
 Platt T, Gallegos CL, Harrison WG (1980) Photoinhibition of
photosynthesis in natural assemblages of marine phytoplankton. J
Mar Res 38: 687-701
 Rappaport F, Béal D, Joliot A, Joliot P (2007) On the advantages
of using green light to study fluorescence yield changes in leaves.
Biochim Biophys Acta 1767: 56–65
 Schreiber U (2004) Pulse-amplitude-modulation (PAM)
fluorometry and saturation pulse method: an overview. In:
Papageorgiou GC, Govindjee (eds.) Chlorophyll a Fluorescence: A
Signature of Photosynthesis. Springer, The Netherlands, pp. 279-319
 Schreiber U, Bilger W, Neubauer C (1994) Chlorophyll
fluorescence as a non-intrusive indicator for rapid assessment of in
vivo photosynthesis. Ecological studies: analysis and synthesis
(USA) 100: 49-70
 Schreiber U, Klughammer C (2013) Wavelength-dependent
photodamage to Chlorella investigated with a new type of multicolor PAM chlorophyll fluorometer. Photosynthesis Research 114:
165-177
 Schreiber U, Klughammer C, Kolbowski J (2011) High-end
chlorophyll fluorescence analysis with the MULTI-COLOR-PAM. I.
Various light qualities and their applications. PAN (2011) 1: 1-19
http://www.walz.com/downloads/pan/PAN11001.pdf
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 Schreiber U, Schliwa U, Bilger W (1986) Continuous recording
of photochemical and non-photochemical chlorophyll fluorescence
quenching with a new type of modulation fluorometer. Photosynth
Res 10: 51–62

van Kooten O, Snel J (1990) The use of chlorophyll
fluorescence nomenclature in plant stress physiology. Photosynth
Res 25: 147–150
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10
INDEX
Index
A E Actinic Light 3, 33, 46, 66, 76, 86, 98 AL + Y 34 AL Time 38 alpha 57, 61 Auto MF‐High 45 Auto‐Gain 37 Averaging 76 Eilers and Peeters model 57 Elements for system operation 33 Emission Spectra 7 EP Model 57 ETR 42 57, 61, 63 ETRmax F B battery 10, 47, 102, 105 Battery Charger MINI‐PAM/L 10 C cardinal points of light curve 57 channels‐window 39 Channels‐window 39 chlorophyll calibration 73 chlorophyll content 42, 69, 71, 72, 73 Chlorophyll content 36 chlorophyll content manual correction 73 clock 36, 56, 97 COB LED‐Array 6 Compact Version 5 D data‐directories deconvolution Default‐button Definitions and Equations 112
11 42, 70 46 23 Far Red light fast kinetic window File Fit button fit parameter window fluo spec window Fluorescence Levels fluorescence parameters Fluorescence Ratio Parameters Fo, Fm, Fo´, Fm´, and F Fo, Fm‐determination Fo,Fm Fo´ calculation Fo-I1 Rise Analysis Ft 39 Ft values Fv/Fm and Y(II) Fv/Fm Definition 46 75 85 60 86 74 23 49 25 23 34 52 25 81 49 25 25 G Gain Genty grid 37 28 86 CHAPTER10
INDEX
Measuring Light 33, 44, 62, 101 measuring light intensity 45 H help 86 N I Ik 61 Ik Value indicator lamp info 57 34 86 N(F) New Record New Report noise NPQ Definition J join 41 37, 52, 65, 96 48 10, 41, 76 25 O 86 O‐I1 Overload 75 37 K Konz= L LC Fit Parameters 60 lcp files 56 light curve edit 53 Light Curve EP 57 light curve fit parameters 56 Light curve fit parameters 86 light curve illumination steps 55 Light Curve Platt et al. 59 light curve termination 54 light curve window 51 Light Sensor 9, 14, 103 M main menu Meas. Freq. MEASURE P 73 85 45 36, 65 PAR lists 86, 89 PAR Lists 13 Photomultiplier 38 Photomultiplier activation button 38 Phyto.cfg 11 Platt et al. model 59 87 PPS2/PPPS printer 85 Q qL Definition qN and NPQ qN Definition qP and qL qP Definition 25 28 25 27 25 R Rapid Light Curve 57 113
CHAPTER 10
INDEX
Record 65 record view 66 Ref6 files 69 Reference Excitation Spectra 21, 42, 66, 68, 69, 72, 74 reference spectra generation 72 reference window 68 rel ETR 87 Report 64 report view 65 report window 64 Computer Requirements ST 34, 75 Stirrer Stirring Device WATER‐S 114
46 9 T tooltips 86 U US‐SQS/WB S Safety 1 Sat Pulse settings 47 SAT‐Pulse 35 Saturation Pulse Analyses 23 Saturation Pulse Analysis 24 Script File Commands 93 Script files 36, 90 settings in report 66 settings‐window 44 Sigma(II) 78 single turnover saturating flash 75 slow kinetics window 49 Slow Kinetiks start 49 software installation 11 Specification Battery Charger MINI-PAM/L
103 103 9, 14, 103 V VIEW View Pulse 37, 65 35, 48 W Warranty 115 Y Y(II) Definition Y(NO) Definition Y(NO), Y(NPQ) and Y(II) Y(NPQ) Definition 25 25 28 25 Z Zoff 40 CHAPTER 11
11
WARRANTY
Warranty
All products supplied by the Heinz Walz GmbH, Germany, are
warranted by Heinz Walz GmbH, Germany to be free from
defects in material and workmanship for two (2) years from the
shipping date (date on invoice).
11.1
Conditions
This warranty applies if the defects are called to the attention
of Heinz Walz GmbH, Germany, in writing within two (2) years
of the shipping date of the product.
This warranty shall not apply to
-
any defects or damage directly or indirectly caused by
or resulting from the use of unauthorized replacement
parts and/or service performed by unauthorized
personnel.
-
any product supplied by the Heinz Walz GmbH,
Germany which has been subjected to misuse, abuse,
abnormal use, negligence, alteration or accident.
-
to damage caused from improper packaging during
shipment or any natural acts of God.
-
to batteries, cables, calibrations, fiberoptics, fuses, gas
filters, lamps, thermocouples, and underwater cables.
Submersible parts of the DIVING-PAM or the underwater
version of the MONITORING-PAM have been tested to be
watertight down to the maximum operating depth indicated in
the respective manual. Warranty shall not apply for diving
depths exceeding the maximum operating depth. Further,
warranty shall not apply for damage resulting from improper
115
CHAPTER 11
WARRANTY
operation of devices, in particular, the failure to properly seal
ports or sockets.
11.2
Instructions
To obtain warranty service, please follow the
instructions below:
-
The Warranty Registration form must be completed
and returned to Heinz Walz GmbH, Germany.
-
The product must be returned to Heinz Walz
GmbH, Germany, within 30 days after Heinz Walz
GmbH, Germany has received written notice of the
defect. Postage, insurance, and/or shipping costs
incurred in returning equipment for warranty
service are at customer expense. Duty and taxes
are covered by Walz. Accompany shipment by the
Walz Service and Repair form available at:
http://www.walz.com/support/repair_service.html
116
-
All products being returned for warranty service
must be carefully packed and sent freight prepaid.
-
Heinz Walz GmbH, Germany is not responsible or
liable, for missing components or damage to the
unit caused by handling during shipping. All claims
or damage should be directed to the shipping
carrier.